Integrated sample-processing system

ABSTRACT

The invention provides an integrated sample-processing system and components thereof for preparing and/or analyzing samples. The components may include a transport module, a fluidics module, and an analysis module, among others.

CROSS-REFERENCES

This application is a continuation of PCT Patent Application Ser. No.PCT/US00/12277, filed May 3, 2000, which is incorporated herein byreference.

This application is based upon and claims priority under 35 U.S.C. §119from the following U.S. Provisional Patent Applications, each of whichis incorporated herein by reference: Ser. No. 60/132,262, filed May 3,1999; Ser. No. 60/132,263, filed May 3, 1999; Ser. No. 60/138,737, filedJun. 11, 1999; Ser. No. 60/138,893, filed Jun. 11, 1999; Ser. No.60/153,251, filed Sep. 10, 1999; and Ser. No. 60/167,301, filed Nov. 24,1999.

This application incorporates by reference the following U.S. patentapplications: Ser. No. 08/929,095, filed Sep. 15, 1997, Ser. No.09/062,472, filed Apr. 17, 1998; Ser. No. 09/160,533, filed Sep. 24,1998; Ser. No. 09/349,733, filed Jul. 8, 1999; Ser. No. 09/468,440,filed Dec. 21, 1999; Ser. No. 09/478,819, filed Jan. 5, 2000; Ser. No.09/494,407, filed Jan. 28, 2000; and Ser. No. 09/556,030, filed Apr. 20,2000.

This application also incorporates by reference the following PCT patentapplications: Ser. No PCT/US99/01656, filed Jan. 25, 1999; Ser. No.PCT/US99/03678, filed Feb. 19, 1999; Ser. No. PCT/US99/08410, filed Apr.16, 1999; Ser. No. PCT/US99/16057, filed Jul. 15, 1999; Ser. No.PCT/US99/16453, filed Jul. 21, 1999; Ser. No. PCT/US99/16621, filed Jul.23, 1999; Ser. No. PCT/US99/16286, filed Jul. 26, 1999; Ser. No.PCT/US99/16287, filed Jul. 26, 1999; Ser. No. PCT/US99/24707, filed Oct.19, 1999; Ser. No. PCT/US00/00895, filed Jan. 14, 2000; Ser. No.PCT/US00/03589, filed Feb. 11, 2000; Ser. No. PCT/US00/04543, filed Feb.22, 2000, and Ser. No. PCT/US00/06841, filed Mar. 15, 2000.

This application also incorporates by reference the following U.S.provisional patent applications: Ser. No. 60/138,311, filed Jun. 9,1999; Ser. No. 60/138,438, filed Jun. 10, 1999; Ser. No. 60/142,721,filed Jul. 7, 1999; Ser. No. 60/164,633, filed Nov. 10, 1999;60/165,813, filed Nov. 16, 1999; Ser. No. 60/167,463, filed Nov. 24;1999; Ser. No. 60/178,026, filed Jan. 26, 2000; Ser. No. 60/182,036,filed Feb. 11, 2000; Ser. No. 60/182,419, filed Feb. 14, 2000; Ser. No.60/190,265, filed Mar. 17, 2000; Ser. No. 60/191,890, filed Mar. 23,2000; Ser. No. 60/193,586, filed Mar. 30, 2000; Ser. No. 60/197,324,filed Apr. 14, 2000; Ser. No. 60/200,530, filed Apr. 27, 2000, and Ser.No. 60/200,594, filed Apr. 28, 2000.

This application also incorporates by reference the followingpublications: K. E. van Holde, Physical Biochemistry (2^(nd) ed. 1985);William Bains, Biotechnology from A to Z (1993); Richard P. Haugland,Handbook of Fluorescent Probes and Research Chemicals (6^(th) ed. 1996);Joseph R. Lakowicz, Principles of Fluorescence Spectroscopy (2^(nd) ed.1999). Bob Sinclair, Everything's Great When It Sits on a Chip: A BrightFuture for DNA Arrays, 13 THE SCIENTIST, May 24, 1999, at 18; andCharles R. Cantor and Paul R. Schimmel, Biophysical Chemistry (1980).

FIELD OF THE INVENTION

The invention relates to sample-processing systems, and moreparticularly to integrated sample-processing systems and componentsthereof for preparing and/or analyzing samples.

BACKGROUND

Modern laboratory techniques such as high-throughput screening ofcandidate drug compounds may involve preparing and analyzing hundreds ofthousands or millions of samples. Recently, the processing of suchsamples has been facilitated by packaging samples in high-density sampleholders, such as microplates, for analysis together in an automateddevice. FIG. 1 shows an offset stack of microplates, illustrating therange in possible well densities and well dimensions. Plate 130 has 96sample wells. Plate 132 has 384 wells. Plate 134 has 1536 wells. Plate136 has 3456 wells. Plate 138 has 9600 wells.

Unfortunately, prior systems for processing large numbers of sampleshave significant shortcomings. For example, prior systems may not havethe flexibility to process sample holders with different sampledensities, or the sensitivity or accuracy to process sample holders withvery small samples. Moreover, prior systems may be limited to single(unit) operations, meaning, for example, that they can dispense samplesor analyze samples, but not do both. Thus, prior systems may requiredifferent apparatus for different applications, or lead to missed hits,limited research capabilities, lower throughput, and/or increased costsfor compounds, assays, and reagents.

SUMMARY OF THE INVENTION

The invention provides an integrated sample-processing system andcomponents thereof for preparing and/or analyzing samples. Thecomponents may include a transport module, a fluidics module, and ananalysis module, among others.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a top view of overlapping microplates showing variations inwell size and well density.

FIG. 2 is a perspective view of a system for preparing and/or analyzingsamples.

FIG. 3 is a perspective view of an alternative system for preparingand/or analyzing samples.

FIG. 4 is a schematic view of a generalized system for preparing and/oranalyzing samples.

FIG. 5 is a top view of a 96-well microplate constructed in accordancewith aspects of the invention.

FIG. 6 is a cross-sectional view of the microplate in FIG. 5, takengenerally along line 6—6 in FIG. 5.

FIG. 7 is a first enlarged portion of the cross-sectional view in FIG.6, showing details of a sample well.

FIG. 8 is a second enlarged portion of the cross-sectional view in FIG.6, showing details of a reference fiducial.

FIG. 9 is a top view of a 384-well microplate constructed in accordancewith the invention.

FIG. 10 is a cross-sectional view of the microplate in FIG. 8, takengenerally along line 10—10 in FIG. 9.

FIG. 11 is an enlarged portion of the cross-sectional view in FIG. 10,showing details of a sample well.

FIG. 12 is an enlarged cross-sectional view of the microplate in FIG. 9,taken generally along line 12—12 in FIG. 9, showing details of areference fiducial.

FIG. 13 is a perspective view of a 1536-well microplate constructed inaccordance with the invention.

FIG. 14 is a top view of the microplate in FIG. 13.

FIG. 15 is an enlarged portion of the top view in FIG. 14, showingdetails of the sample wells.

FIG. 16 is a cross-sectional view of the microplate in FIG. 14, takengenerally along line 16—16 in FIG. 14.

FIG. 17 is an enlarged portion of the cross-sectional view in FIG. 16,showing details of the sample wells.

FIG. 18 is a partial perspective view of a transport module constructedin accordance with aspects of the invention.

FIG. 19 is a partially exploded perspective view of a latch mechanismfrom the transport module of FIG. 18.

FIG. 20 is a cross-sectional view of the latch mechanism of FIG. 19,taken generally along line 20—20 in FIG. 19, showing the latch mechanismin use supporting a stack of plates.

FIG. 21 is a multi-panel time-lapse cross-sectional view of the latchmechanism of FIG. 20, showing the latch mechanism in use to input aplate from a stack of plates.

FIG. 22 is a multi-panel time-lapse cross-sectional view of the latchmechanism of FIG. 20, showing the latch mechanism in use to output aplate to a stack of plates.

FIG. 23 is a partially exploded perspective view of an intrasite driverfrom the transport module of FIG. 18.

FIG. 24 is a partially exploded perspective view of an intersite driverfrom the transport module of FIG. 18.

FIG. 25 is a partially schematic view of a noncontact fluid dispenser,showing a positive-displacement syringe pump with sapphire-tippeddispense elements.

FIG. 26 is a partially schematic view of an alternative noncontact fluiddispenser, showing a positive-displacement syringe pump with solenoidvalves and sapphire-tipped dispense elements.

FIG. 27 is a partially schematic view of another alternative noncontactfluid dispenser, showing a positive-pressure pump with solenoid valvesand sapphire-tipped dispense elements.

FIG. 28 is a schematic view of yet another alternative noncontact fluiddispenser, showing the relationship between fluid reservoirs, pumps, anddispense elements for the fluid dispenser shown (together with atransport module and an analysis module) in FIG. 2 as part of anintegrated system for preparing and/or analyzing samples.

FIG. 29 is a perspective view of a dispense driver for the fluiddispenser of FIG. 28, showing the relationship between the dispenseelements and a sample holder. The view is an enlargement of componentsshown in FIG. 18 in connection with a transport module.

FIG. 30 is a front view of a portion of the fluid dispenser of FIG. 28,showing a fluid control unit including a bank of associated syringepumps.

FIG. 31 is a front view of a bank of dispense elements for the fluiddispenser of FIG. 28.

FIG. 32 is an exploded perspective view of the bank of dispense elementsshown in FIG. 31.

FIG. 33 is a cross-sectional view of a portion of the bank of dispenseelements shown in FIG. 32, taken generally along line 33—33 in FIG. 32.

FIG. 34 is a six-panel, time-lapse schematic view of a single pin from apin transfer device, showing how the device may be used to transferfluid.

FIG. 35 is a schematic view of a pin transfer device and associatedsample holder, showing shortcomings associated with a rigid array ofpins.

FIG. 36 is a perspective view of an alternative pin transfer device andassociated sample holder.

FIG. 37 is a cross-sectional view of the alternative pin transfer deviceand associated sample holder of FIG. 36, taken generally along line37—37 in FIG. 36.

FIG. 38 is a partially schematic top view of a variable-pitch-arrayfluid dispenser, showing the dispenser in use with a microplate.

FIG. 39 is a partially schematic side view of the variable-pitch-arrayfluid dispenser and microplate of FIG. 38, taken generally along line39—39 in FIG. 38.

FIG. 40 is a perspective view of a microplate sealing system constructedin accordance with aspects of the invention.

FIG. 41 is a perspective view of a sealed microplate.

FIG. 42 is a top view of a microplate sealing system, shown applyingsheets to a microplate.

FIG. 43 is a top view of a microplate sealing system, shown removingsheets from a microplate.

FIG. 44 is a schematic view of a system for controlling ambientconditions around samples contained in wells in a stack of microplatesin accordance with aspects of the invention.

FIG. 45 is a side view of a microplate having an aperture in the base toallow thermal and gas circulation below sample wells contained in themicroplate and projections for supporting another microplate.

FIG. 46 is a perspective view of portions of two alternative embodimentsof a lid-spacer for separating stacked microplates.

FIG. 47 is a side view of alternated stacked microplates and lid-spacersbeing manipulated by singulation latches.

FIG. 48 is a schematic view of a sample processing system utilizinglid-spacing devices in an incubating station.

FIG. 49 is a partially exploded perspective view of a system foranalyzing samples in accordance with aspects of the invention, showing atransport module and an analysis module.

FIG. 50 is a schematic view of an optical system from the analysismodule of FIG. 49.

FIG. 51 is a partially schematic perspective view of the apparatus ofFIG. 50.

FIG. 52 is a schematic view of photoluminescence optical components fromthe apparatus of FIG. 50.

FIG. 53 is a schematic view of chemiluminescence optical components fromthe apparatus of FIG. 50.

FIG. 54 is a top perspective view of a portion of a transport mechanismfrom the analysis module of FIG. 49.

FIG. 55 is a bottom perspective view of the portion of a transportmechanism shown in FIG. 54.

FIG. 56 is a partial cross-sectional view of the portion of a transportmechanism shown in FIGS. 54 and 55, taken generally along the line 56—56in FIG. 55.

FIG. 57 is a perspective view of a base platform and associated drivemechanisms for moving the portion of a transport mechanism shown inFIGS. 54—56 along X and Y axes relative to the base platform.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 2 is a perspective view of a system 500 for preparing and/oranalyzing samples. System 500 includes at least one input/output (I/O)site 502 for sample input and output, and a plurality of functionmodules (or stations) for performing a plurality of functions, includinga transport module 504, a fluidics module 506, and an analysis module508. The transport module participates in sample transport, for example,by shuttling a sample or sample holder between the I/O sites, fluidicsmodule, and analysis module. The fluidics module participates in samplepreparation, for example, by adding (and/or removing) a component of asample to a sample holder. The analysis module participates in sampleanalysis, for example, by performing an optical analysis of a samplebased on photoluminescence, chemiluminescence, absorbance, andscattering, among others. The components of system 500 may be configuredto enhance function, convenience, and/or appearance.

FIG. 3 is a perspective view of an alternative system 600 for preparingand/or analyzing samples. System 600 resembles system 500 and similarlyincludes at least one I/O site 602, a transport module 604, a fluidicsmodule 606, and an analysis module 608. However, in system 600, the I/Osites include processing bins 610 to facilitate handling multiple sampleholders. Moreover, the fluidics module and the transport and analysismodules are positioned on different shelves of a moveable multi-tieredcart 612, enhancing portability and reducing footprint (<1 squaremeter).

FIG. 4 is a schematic view of a generalized system 700 for preparingand/or analyzing samples. System 700 includes at least one I/O site 702and a plurality of function modules, including a transport module 704, afluidics module 706, and an analysis module 708, as above, as well as Nauxiliary modules 710 associated with redundant and/or additionalfunctionalities, such as cleaning, sealing, storage, sample preparation,etc. Here, N may range from zero to several or more. A cleaning modulemight include components for emptying and/or cleaning sample holders. Asealing module might include components for sealing, unsealing, and/orotherwise covering and uncovering sample holders. An incubation modulemight include components for incubating sample holders and theirassociated samples, with environmental control of atmosphere,temperature, agitation, and so on. A sample preparation module mightinclude components for particular sample-preparation functions, such asa thermocycler for performing heating and cooling during the polymerasechain reaction (PCR).

Function modules generally include one or more function sites at which acorresponding function is performed. For example, a fluidics module mayinclude a dispense site 712 at which a fluid is dispensed, an analysismodule may include an examination (“exam”) site 714 at which a sample isanalyzed, and an auxiliary module may include an auxiliary site 716 atwhich an auxiliary function is performed, such as cleaning, sealing,storage, etc. A transport module may be connected directly or indirectlywith I/O sites 702 for sample input and output, and with one or more ofthe function sites. If the transport module is connected indirectly to afunction (or I/O) site, the transport module might hand off a sampleholder at a transfer site to a separate transport mechanism associatedwith the respective function module. A transport module also may beconnected to additional robotics for providing and removing sampleholders from the I/O sites.

System 700 generally may include any desired combination of functionmodules. For example, a simple system may include a pair of modules,such as a fluidics module and a transport module, or an analysis moduleand a transport module. These systems might be used to prepare a sampleor analyze a sample, respectively. A more complex system may includeseveral modules, such as a fluidics module, an analysis module, anincubation module, and a transport module. This more complex systemmight be used to prepare a sample, analyze a sample, or both prepare andanalyze a sample, for example, by adding a reporter to the sample usingthe fluidics module, incubating the sample using the incubation module,reading the sample using the analysis module, and inputting, outputting,and transporting the sample using the transport module.

The function modules generally may be accessed in any desired order. Forexample, a sample might be analyzed only after fluid dispensing, or bothbefore and after fluid dispensing if a multi-step assay is beingperformed and/or if a background is being subtracted. The order ofaccess may be controlled using a controller that may schedule andinitiate singulation of samples to and from I/O sites, transport betweensites, dispensing at a dispensing site, and/or analysis at an analysissite. The order and timing of such movements will depend on the natureof the assay and generally will differ for kinetics assays (where timingis crucial) and endpoint assays (where timing is not crucial, so long asan endpoint has been reached).

The function modules generally may be combined or integrated in anydesired way. For example, a single module may perform fluidics andcleaning operations, and a single transport mechanism may access any orall of the I/O and/or function sites.

Further aspects of the invention are presented in the followingsections: (A) sample holders, (B) transport module, (C) fluidics module,(D) auxiliary modules, (E) analysis module; and (F) additional examples.

A. Sample Holders

The system and its components may be used with a variety of sampleholders and sample holder features. As used here, “sample holder”generally comprises any substrate or material capable of supporting asample so that the sample holder and associated sample can betransported by an automatic transport module and subjected to a functionsuch as fluid dispensing or optical analysis at a corresponding functionmodule. Sample holders may be used alone, in stacks, or in combinationwith seals or covers, as described below. Sample holders may supportsamples at low, intermediate, or high density, and be designed forsingle or multiple use.

Exemplary sample holders include microplates, PCR plates, biochips, andchromatography plates, among others. A microplate is a multi-well sampleholder, typically but not exclusively used for luminescenceapplications. Preferred microplates are described below. A PCR plate isa multi-well sample holder used for performing PCR. Preferred PCR plateswould include a footprint, well spacing, and well shape similar to thoseof the preferred microplates, while possessing a stiffness adequate forautomated handling and a thermal stability adequate for PCR. A biochipis a small, flat surface (such as a glass or silicon wafer) onto whichbiomolecules (such as nucleic acids and proteins) are immobilized indistinct spots or arrays. Biochips include DNA chips, DNA microarrays,gene arrays, and gene chips, among others. Preferred biochips aredescribed in Bob Sinclair, Everything's Great When It Sits on a Chip: ABright Future for DNA Arrays, 13 THE SCIENTIST, May 24, 1999, at 18. Achromatography plate is a flat surface used for performingchromatography, such as thin-layer chromatography.

Microplates are a preferred sample holder, and the system and itscomponents may be designed for use with microplates having some or allof the following features. For example, suitable microplates may includemicroplates having any number of wells, including 96, 384, and 1536,among others. Suitable microplates also may include microplates havingwells with elevated bottoms, frusto-conical shapes, and/or low volumes,or wells configured to reduce the formation and/or trapping of bubbles,as described in the following U.S. patent applications, which areincorporated herein by reference: Ser. No. 08/840,553, filed Apr. 14,1997; and Ser. No. 09/478,819, filed Jan. 5, 2000. Suitable microplatesalso may include microplates having reference fiducials in or around aperimeter portion of the microplate (or elsewhere), as described in PCTPatent Application Ser. No. PCT/US99/08410, filed Apr. 16, 1999, whichis incorporated herein by reference. Such reference fiducials may bemolded into the plate and/or applied to the plate by silkscreen, colortransfer, hot stamping, and/or application of reflective paint, amongothers. Suitable microplates also may include microplates having abarcode for reading by a barcode reader, as described in U.S. patentapplication Ser. No. 09/160,533, filed Sep. 24, 1998, which isincorporated herein by reference. Suitable microplates also may beuseable in combination with seals for sealing individual wells in amicroplate, and/or spacer members for separating individual microplatesin a stack, as described below.

FIGS. 5-17 show a set of preferred microplates that have similar heightsand footprints but that differ in well shape, well size, and/or welldensity. These microplates include (1) 96-well microplates, (2) 384-wellmicroplates, (3) 1536-well microplates, and (4) miscellaneousmicroplates.

1. 96-Well Microplates

FIG. 5 is a top view of a 96-well microplate 1200 constructed inaccordance with aspects of the invention. Microplate 1200 includes aframe 1202 and a plurality of sample wells 1204 disposed in the frame.In some embodiments, microplate 1200 may include one or more referencefiducials 1206 disposed in the frame.

Frame 1202 is the main structural component of microplate 1200. Theframe may have various shapes and various dimensions. In microplate1200, frame 1202 is substantially rectangular, with a major dimension Xof about 127.8 mm and a minor dimension Y of about 85.5 mm. Tolerancesin plate dimensions typically are about ±0.5-1.0 mm for polystyreneplates, but may increase to about ±2 mm for polypropylene plates,especially if the polypropylene plates are produced using molds designedfor polystyrene plates. Frame 1202 may be adapted for ease of use andmanufacture. For example, frame 1202 may include a base 1208 tofacilitate handling and/or stacking, and frame 1202 may include notches1210 to facilitate receiving a protective lid. Frame 1202 may beconstructed of a material, such as a thermoplastic, that is sturdyenough for repeated, rugged use and yet minimally photoluminescent toreduce background upon illumination.

Frame 1202 includes a sample well region 1212 and an edge region 1214forming a perimeter 1216 around the sample well region. Sample wells maybe disposed in the sample well region in various configurations. Inmicroplate 1200, sample wells 1204 are disposed in sample well region1212 in a substantially rectangular 8×12 array, with a pitch (i.e.,center-to-center interwell spacing) along both X and Y of about 9 mm.This pitch corresponds to a density of wells of about one well per 81 mm².

Reference fiducials 1206 may be used for identification, alignment,and/or calibration of the microplate. Reference fiducials may bedisposed in the sample well region and/or the edge region in variousconfigurations. In microplate 1200, reference fiducials 1206 aredisposed in edge region 1214, substantially aligned with a row of samplewells along the X dimension, although reference fiducials also may beoffset from the rows of sample wells. Reference fiducials preferentiallyare positioned in comers of the microplate, near where optical analysisbegins, so that they may quickly be identified and analyzed. Referencefiducials may be positioned in rotationally symmetric positions, so thatmicroplates may be loaded into an optical device and analyzed backwardswithout difficulty. Alternatively, reference fiducials may be positionedin rotationally asymmetric positions, so that the system can ascertainwhich way the microplate is oriented; information on orientation isuseful because samples typically are not positioned symmetrically withinthe microplate. Further aspects of reference fiducials are described inPCT Patent Application Ser. No. PCT/US99/08410, filed Apr. 16, 1999,which is incorporated herein by reference.

FIG. 6 is a cross-sectional view of microplate 1200, showing samplewells 1204, reference fiducial 1206, and base 1208. In microplate 1200,frame 1202 has a top 1218, a substantially parallel bottom 1220, andsubstantially perpendicular sides 1222. Top 1218 may have variousshapes, although it typically is flat. (Top 1218 may be surrounded by araised edge to facilitate stacking.) Frame 1202 has a height H of about12 mm, corresponding generally to the separation between top 1218 andbottom 1220. Tolerances in plate height typically are about 0.5 mm orless. Sample wells 1204 are disposed with open, optically transparentends 1224 directed toward top 1218, and closed, optically opaque ends1226 directed toward bottom 1220. In some embodiments, optically opaqueends 1226 may be replaced by optically transparent ends to permit bottomillumination and/or detection. Reference fiducial 1206 is disposed ontop 1218, although reference fiducials also may be disposed on bottom1220 and/or sides 1222.

The preferred plate height is determined by a variety of considerations.Generally, taller plates with elevated bottoms and/or filled wells putthe samples closer to the detector for analysis, increasing numericalaperture and hence signal. Conversely, shorter plates allow more platesto be stacked into processing bins for longer periods of unattendedoperation. The specified height of about 12 mm generally is large enoughto facilitate handling by sample handlers and/or a stage, and yet smallenough to permit optical analysis of the entire well. Moreover, thespecified height generally is sufficient to ensure that the microplatesare sufficiently flat for analysis.

FIG. 7 is a first enlarged portion of the cross-sectional view in FIG.6, showing details of sample wells 1204. Sample wells may have variousshapes and various dimensions, as described in detail in subsequentsections. In microplate 1200, sample wells 1204 are substantiallyfrusto-conical, with substantially straight side walls 1228 and asubstantially flat bottom wall 1230. In microplate 1200, opticallyopaque ends 1226 are positioned about 6.7 mm below top 1218, and about5.3 mm above bottom 1220. Sample well 1204 is characterized by a topdiameter D_(T,96), a bottom diameter D_(B,96), a height H₉₆, and a coneangle θ₉₆. Here, θ₉₆ is the included angle between side walls 1228. Inmicroplate 1200, D_(T,96) is about 4.5 mm, D_(B,96) is about 1.5 mm, H₉₆is about 6.7 mm, and θ₉₆ is about 25.4°. Sample well 1204 has a totalvolume of about 50 μL, and a smallest practical working volume of about1-40 μL.

FIG. 8 is a second enlarged portion of the cross-sectional view in FIG.6, showing details of reference fiducial 1206. Reference fiducials mayhave various shapes and various dimensions, as described in detail insubsequent sections. In microplate 1200, reference fiducial 1206 issubstantially frusto-conical, with substantially straight side walls1232 and a substantially flat bottom wall 1234. Reference fiducial 1206is characterized by a top diameter D_(T,RF,96), a bottom diameterD_(B,RF,96), a height H_(RF,96), and a cone angle θ_(RF,96). Here,D_(B,RF,96) and θ_(RF,96) are substantially equal to D_(B,96) and θ₉₆,the corresponding values for sample well 1204. H₉₆ is about 1 mm, andD_(T,RF,96) is specified by the other parameters. In some applications,the reference fiducial may contain a luminescent material or solution sothat it is easier to locate. In other applications, the referencefiducial may be used as a blank for determining background, or as anadditional sample well for holding an additional sample. In theseapplications, the reference fiducial may be located and/or analyzedusing the same optical system used to analyze samples in conventionalsample wells.

2. 384-Well Microplates

FIGS. 9-12 are views of a 384-well microplate 1300 constructed inaccordance with aspects of the invention. Microplate 1300 is similar inmany respects to microplate 1200 and includes a frame 1302 and aplurality of sample wells 1304 disposed in a sample well region 1312 ofthe frame. In some embodiments, microplate 1300 may include one or morereference fiducials 1306 disposed in an edge region 1314 or other regionof the frame.

The external dimensions of microplate 1300 are similar to the externaldimensions of microplate 1200. However, the density of sample wells inmicroplate 1300 is four times higher than the density of sample wells inmicroplate 1200. Consequently, the pitch (i.e., the center-to-centerinterwell spacing) in microplate 1300 is about 4.5 mm, or about one-halfthe pitch in microplate 1200. This pitch corresponds to a density ofwells of about four wells per 81 mm². In microplate 1300, referencefiducial 1306 is positioned about midway between two rows of samplewells along the X direction; in contrast, in microplate 1200, referencefiducial 1206 is positioned about in line with a row of sample wellsalong the X direction. This is because the reference fiducials arepositioned in approximately the same position in each microplate, butthe center line of one row of sample wells in microplate 1200 becausethe center line between two rows of sample wells in microplate 1300 asthe density of wells is quadrupled.

Sample wells 1304 in microplate 1300 are similar to sample wells 1204 inmicroplate 1200. Sample wells 1304 may be characterized by a topdiameter D_(T,384), a bottom diameter D_(B,384), a height H₃₈₄, and acone angle θ₃₈₄. The preferred values of D_(B,384) and θ₃₈₄ formicroplate 1300 are substantially similar to the preferred values ofD_(B,96) and θ₉₆ for microplate 1200. However, the preferred value forD_(T,384), which is about 4.7 mm, is smaller than the preferred valuefor D_(T,384), which is about 6.7 mm. In microplate 1300, the upperdiameter must be smaller than the upper diameter of the sample wells inmicroplate 1200, because the sample wells are close packed, leaving nomore interwell spacing than necessary for moldability. In turn, thepreferred value for H₃₈₄ is about 4.7 mm, so that the wells are elevatedby about 7.3 mm. Sample well 1304 has a total volume of about 25 μL, anda smallest practical working volume of about 1-12 μL.

Reference fiducial 1306 in microplate 1300 may be essentially identicalto reference fiducial 1206 in microplate 1200.

3. 1536-Well Microplates

FIGS. 13-17 are views of a 1536-well microplate 1350 constructed inaccordance with aspects of the invention. Microplate 1350 is similar inmany respects to microplates 1200 and 1300, and includes a frame 1352and a plurality of sample wells 1354 disposed in the frame. The pitch inmicroplate 1350 is about 2.25 mm, or about one-half the pitch inmicroplate 1300 and about one-fourth the pitch in microplate 1200. Thispitch corresponds to a density of wells of about sixteen wells per 81mm².

Sample wells 1354 may be exclusively frusto-conical, like sample wells1204 in microplate 1200 and sample wells 1304 in microplate 1300.However, due to spatial constraints, the volume of such wells would haveto be small, about 1-2 μL. Smaller wells are easier to mold and keepwithin tolerances, but they provide less flexibility and place morestringent demands on fluid dispensing and analytical equipment.Alternatively, sample wells 1354 may have a frusto-conical lower portion1306 coupled to a cylindrical upper portion 1308. The volume of suchwells may be larger, for example, about 7-8 μL. Larger wells are moredifficult to mold, but they permit use of a wider range of samplevolumes and therefor a wider range of assay formats. Larger samplevolumes may be useful if the microplate is used in conjunction withstandard fluid dispensing equipment, because the standard equipment mayhave difficulty dispensing small volumes. Larger sample volumes also maybe useful if reagents are to be added to the well from stock solutions,such as 100×DMSO or DMF stock solutions, because they make it lessnecessary to dispense very tiny amounts of stock solution to obtainadequate dilution. Larger sample volumes also may be useful forcell-based assays, because cells may live longer in a larger volume ofmedium.

Reference fiducials in microplate 1350 may be essentially identical toreference fiducials 1206 in microplate 1200 and reference fiducials 1306in microplate 1300. However, reference fiducials in microplate 1350 maybe more important than reference fiducials in plates 1200 and 1300because the well dimensions in microplate 1350 may approach the moldingtolerances, making it more likely that wells will be significantlydisplaced from their nominal positions.

4. Miscellaneous Microplates

The system and its components also may be designed for use with some orall of the following microplates:

-   (a) A microplate having a frame portion and a top portion, where an    array of wells is formed in the top portion. The wells are organized    in a density of at least about 4 wells per 81 mm². Each well has a    bottom wall that is elevated at least about 7 millimeters above a    plane defined by a bottom edge of the frame.-   (b) A microplate having an array of conical wells organized in a    density of at least about 4 wells per 81 mm².-   (c) A microplate having an array of conical wells, where each well    has a maximum volume capacity of less than about 55 microliters. A    preferred small-volume well design has a volume capacity of 1-20    microliters.-   (d) A microplate having an array of wells in the top portion, where    each well has a maximum volume capacity of less than about 55    microliters and a well bottom that is elevated at least about 7    millimeters above a plane defined by a bottom edge of the frame.-   (e) A microplate having an array of wells in a top portion,    organized in a density of at 2 least about 4 wells per 81 mm², where    each well has a conical portion characterized by a cone angle of at    least about 8°.-   (f) A microplate having an array of conical wells characterized by a    cone angle θ, where θ=2arcsin (NA/n) and NA is equal to or greater    than about 0.07.-   (g) A microplate having an array of wells organized in a density of    at least about 16 wells per 81 mm², where each well has a    frusto-conical bottom portion and a substantially cylindrical upper    portion.-   (h) A microplate comprising a frame and a plurality of    frusto-conical sample wells disposed in the frame, where the sample    wells are characterized by a cone angle of at least about 8°. The    microplate further may include a reference fiducial that provides    information to facilitate sample analysis.-   (i) A microplate having 864 sample wells, 3456 sample wells, or 9600    sample wells.-   (j) A microplate formed of black, white, or clear material, or a    combination thereof.-   (k) A microplate suitable for performing PCR.

B. Transport Module

FIG. 18 shows a transport module 2100 constructed in accordance withaspects of the invention. The transport module generally comprises anymechanism or system for automatically shuttling a plate or other sampleholder between an I/O site and one or more function or transfer sites.The transport module may enhance convenience by reducing humanintervention and enhance throughput by reducing the time required toprocess multiple samples.

The transport module may include one or more I/O sites 2102, one or morefunction or transfer sites 2104, and mechanisms for moving platesbetween the I/O and function sites. An I/O site is a site at whichsample holders are input and/or output. A function site is a site atwhich one or more functions are performed, such as fluid dispensing,analysis, cleaning, containment, and/or incubation, among others. Atransfer site is a site at which a sample holder is transferred to atransport mechanism (independently) associated with a function module.The mechanisms for moving plates may include a latch mechanism 2106, anintrasite driver 2108, and/or an intersite driver 2110. The latchmechanism may be used for singulating plates from and to stacks ofplates. The intrasite and intersite drivers may be used for movingplates between and within sites, respectively. These latch mechanism anddrivers may share features and/or components, or be substantially ortotally independent.

The transport module also may include other features, such as a barcodereader 2120 for reading an informational barcode optionally associatedwith a plate, a plate sensor for detecting the presence of a plate atone or more sites within the transport module, and one or moreinterfaces for interactions with function modules. The plate sensors maybe positioned at one or more sites and may be configured to accommodatevariations in plate thickness, color, and so on. The interfaces mayinclude mounting sites for a dispense head 2124 and/or a dispense driver2126 associated with a fluidics function module.

The transport module may employ a variety of singulation strategies,depending in part on the number of I/O sites, the nature of the I/Osites (i.e., input, output, or both), and the location in the stack fromwhich plates are taken and/or added (typically bottom and/or top).Transport module 2100 has two I/O sites, from which plates are takenand/or added at the bottom. Typically, but not necessarily, one of thesesites is dedicated to input, and the other is dedicated to output. Toinput a plate, a robot (1) removes a plate from the bottom of an inputstack of plates at the input site, (2) transports the plate to thetransfer site, and (3) transfers the plate at the transfer site to atransport mechanism for an associated function module. To output aplate, the robot (1) takes the plate from the transport mechanism forthe function module at the transfer site, (2) transports the plate tothe output site, and (3) transfers the plate at the output site to thebottom of an output stack of plates at the output site. In transportmodule 2100, these functions are performed by the intersite andintrasite drivers, with preferred throughputs ranging from about 1second per plate to about 5 seconds per plate.

Further aspects of the transport module are described in the followingsections: (1) latch mechanisms, (2) intrasite drivers, (3) intersitedrivers, (4) additional features, and (5) examples.

1. Latch Mechanisms

The latch mechanism generally comprises any mechanism for inputting asingle plate from a stack of plates and/or outputting a single plate toa stack of plates. The following description addresses (a) the actuationmechanism employed by the latch mechanism, and (b) general attributes ofthe latch mechanism.

a. Actuation Mechanism

FIG. 19 shows an exemplary latch mechanism 2200 for used in the I/Osites. Latch mechanism 2200 includes a latch body 2202 and complementarypairs of latches 2204, pivot pins 2206, retaining pins 2208, torsionsprings 2210, and electromagnets 2212. The latch body is an elongatesubstantially rectangular structure that includes an inward-facingrecess 2214 adjacent each end. The latches are elongate structures thatinclude a pivot portion 2216 and a pick portion 2218. The latches arepivotably mounted in recesses 2214 so that the pivot portion is mountedabout the pivot pin and the pick portion is free to pivot through anangle determined by the retaining pin at one extreme and theelectromagnet at the other extreme.

The latch mechanisms generally are used in pairs to support oppositesides of a plate, and each latch mechanism includes two latches tosupport opposite ends of a single side of the plate. As described below,this combination of two lifters and four latches cooperates to singulatesingle plates from and to the bottom of a stack of plates.

FIG. 20 shows latch mechanism 2200 in use to support a stack 2230 ofplates 2232 a,b, which rest atop pick portions 2218 of latches 2204 andgenerally above a lifter 2234. The pick portions are biased inboard oflatch body 2202 and into the cavity 2236 of the corresponding I/O siteto support the plates by the torsion springs (not shown). The latch alsomay be biased toward this position by other mechanisms, includingcounterweights, electromagnets, and other types of springs. The latchalso may be biased toward this position by having a center of gravityabove and inward of pivot pin 2206.

FIG. 21 shows a four-step input cycle for inputting (or singulating) aplate using the transport module and associated latch mechanisms. Platesmay be input from a stack before fluid dispensing and analysis, andafter incubation, among others.

-   -   Step 1. The first input step (Panel A) comprises raising lifters        2234 to elevate a stack 2230 of plates 2232 a,b through contact        of the plates with an upper surface 2240 of the lifters and to        push latches 2204 into a retracted position through contact of        the latches with a side surface 2242 of the lifters. In the        depicted embodiment, the lifters are raised from their resting        height to their maximum height (about 5 mm), after which        electromagnets 2212 behind each latch are energized to hold the        latch in the retracted position. In other embodiments, the        latches may be moved to and/or held in the retracted position at        alternative times and/or by alternative mechanisms, such as a        solenoid-actuated pin. If it is unnecessary to reverse the        function of the latch (for example, because the latch is used        only for input), the latch may be held in the retracted position        by eliminating a notch 2244 in the lifter that otherwise permits        the pick portion of the latch to move under the plates.    -   Step 2. The second input step (Panel B) comprises lowering        lifters 2234 to lower stack of plates 2230. After pick portion        2218 of latch 2204 has passed below a bottom edge 2250 of input        plate 2232 a, the latches are released by de-energizing        electromagnets 2212, so that the pick portion falls against and        then rides along a side 2252 of the plate. The electromagnets        thus control release of the latch without moving parts. The        input plate is the bottommost plate in the stack of plates.        Input-only latches, which lack a top notch, will ride along the        side of the lifter and then fall against and ride along the side        of the plate automatically.    -   Step 3. The third input step (Panel C) comprises further        lowering lifters 2234, with pick portion 2218 of latch 2204        continuing to ride along the side of input plate 2232 a. The        pick portion will follow the contour of the plate, eventually        falling onto the narrower upper section of the plate, where the        pick portion will be positioned below a bottom surface 2260 of        the second to the bottommost plate 2232 b in the stack.    -   Step 4. The fourth input step (Panel D) comprises further        lowering lifters 2234 until input plate 2232 a moves below pick        portion 2218 of latch 2204 and the pick portion contacts bottom        surface 2260 of second to the bottommost plate 2232 b. The latch        thereby catches the next plate, preventing it from dropping,        while the input plate remains on the lifter for further        lowering. Thus, the lifter retains a single plate, and the latch        retains the rest of the original stack of plates. The angle of        the top of the latch may be such that the normal force from the        weight of the stack is directed through pivot pin 2206, so that        no additional moments are induced on the latch. At this point, a        plate has been singulated for further transport to a dispense        site, an analysis site, or an auxiliary site, as desired.

FIG. 22 shows a four-step output cycle for outputting a plate using thetransport module and associated latch mechanism. Plates may be outputafter fluid dispensing and/or analysis, among others. The latchmechanism functions passively as an output latch but actively as aninput latch, in that the electromagnets are not energized during theoutput cycle but are energized for a brief portion of the input cycledown stroke.

-   -   Step 1. The first output step (Panel A) comprises positioning an        output plate 2280 a (i.e., a plate to be output) on lifter 2234        and raising the lifter to elevate the plate so that it is        positioned beneath the bottommost plate 2280 b in a stack of        plates 2282 to which it is to be added or returned. As the        lifters raise the plate, latches 2204 are pushed out of the way        by the outer contour of the plate.    -   Step 2. The second output step (Panel B) comprises further        raising lifter 2234 until a top surface 2290 of output plate        2280 a contacts a bottom surface 2292 of bottommost plate 2280 b        in stack 2282. The new stack is then lifted above latch 2204.    -   Step 3. The third output step (Panel C) comprises lowering        lifter 2234 so that pick portion 2218 of latch 2204 can drop        into notch 2244 in the lifter, thereby positioning itself        beneath the new stack of plates 2282′.

Step 4. The fourth output step (Panel D) comprises further loweringlifter 2234 to its resting position, leaving output plate 2280 a instack 2282′.

Except as noted above, the output cycle generally resembles the inputcycle. The lifter mechanism raises the plate by a fixed amount, therebycausing it to pass the four spring-loaded latches, which retract as theplate is raised by the lifter. Once the bottom of the plate is above thetop of the latch, the latches are released, and a spring on each latchcauses the latch to extend under the plate. The lifter mechanism then islowered, causing the plate to be captured by the now extended latches.The up-stacked plate thus is added to the bottom of the output stack.

b. General Attributes of the Latch Mechanism

The latch mechanism may be configured to have a low inherent sensitivityto the exact size, shape, construction material, and surface finish ofthe plate. For example, the four inwardly sloping, tapered (or angled)latches may cause the stack of plates to self-center within theplate-input area to accommodate both relatively small and large platesizes. Moreover, the mechanism may drop the plate gently onto the lifterwhen the singulation mechanism disengages from the edges of the plate,so that the plate may be lowered to the intrasite driver withoutspilling fluid from the wells. Moreover, if the ends of the latches aresufficiently smooth, the latches should not exert enough frictionalforce on the edges of the plate to cause the plate to tilt or otherwisehang up as the lifter mechanism is lowered and the plate is placed onthe intersite driver. A flexible latch mechanism is important because itfacilitates use of a wider variety of plates and other sample holderswith a reduced likelihood of failure. The depicted mechanism isespecially suited for use with the microplates described above.

The lifters associated with the latch mechanism generally may beactivated using any suitable mechanism. In the depicted embodiment, thelifters are activated using a spring and electromagnet. In alternativeembodiments, the lifters may be activated by gravity. In such analternative embodiment, the latches may pivot on their support pins suchthat their centers of gravity are offset. Consequently, when the liftermechanism is lowered, the latches are activated by gravity to return totheir nonretracted or extended state, thereby preventing the next platesin the stack from dropping as the lifter mechanism is lowered. If theoffset in the center of gravity of the latches is only enough to causethe latches to return to their extended positions, they should pressonly very lightly on the edges of the plate as it drops.

2. Intrasite Drivers

FIG. 23 shows an intrasite driver 2300, which generally comprises anymechanism for moving samples within an I/O and/or function or transfersite, especially in cooperation with a latch or singulation mechanism.Intrasite driver 2300 includes a lift platform 2302 for raising andlowering plates and a drive mechanism 2304 for raising and lowering thelift platform.

Lift platform 2302 includes a base 2306 and sets of lifters 2308 a,bcorresponding to each I/O and/or function site. The lifters generallycomprise any mechanism configured to raise or lower a plate incooperation with a singulation mechanism. Lifters 2308 a for use in theI/O sites are substantially rectangular and include a top notch 2310 forinteraction with a latch, as described above. Lifters 2308 b for use inthe function site are substantially cylindrical and may be used to raiseand lower a plate for transfer to a transport mechanism associated witha function module, as described below.

Drive mechanism 2304 includes a rotary drive motor 2320, an Acme screw2322, a slide 2324, a pair of opposed cam units 2326, and a pair ofopposed guides 2328. These elements cooperate as described below toproduce a lifting force for raising and lowering lift platform 2302 andthus raising and lowering a plate. Here, rotary motion produced by motor2320 is converted to horizontal linear motion by Acme screw 2322, andthe horizontal motion is in turn converted to variable-rate verticalmotion by cam units 2326.

The rotary drive motor and Acme screw together form a linear actuator,which generally comprises any mechanism for producing a linear force ordisplacement, including a positioning table, a rodless cylinder, a robotmodule, an electric thrust cylinder, a pneumatic cylinder, a linearmotor, a linear voice coil, and a solenoid. Here, the role of the rotarydrive motor may be performed by any mechanism capable of producingrotary motion, including a motor, gear motor, gear reducer, manual handcrank, and micrometer, among others. Similarly, the role of the Acmescrew may be performed by any mechanism capable of converting rotarymotion to linear motion. An Acme screw is a preferred mechanism forconverting rotary motion to linear motion because the Acme screwperforms this function using direct sliding friction, which helps tohold the load in position. In contrast, a belt drive or ball screw maypermit a load to back drive due to gravity when no torque is applied tothe motor. The Acme screw is an elongate structure that is connected ata first end through an intermediate pulley system 2330 to drive motor2320 and at a second end to slide 2324.

The cam units 2326 are substantially rectangular and include a top edge2340, a bottom edge 2342, and a pair of opposed side walls 2344. Theside walls include two sloped drive channels 2348, which function as thecams, and a vertical guidance channel 2350. A drive pin 2352 is insertedthrough each drive channel 2348, and a guide pin 2354 is insertedthrough the guide channel 2350. In alternative embodiments, pins andchannels may be replaced with other components, including ridges,bearings, or rollers. Drive pins 2352 inserted into drive channels 2348are connected to slide 2324 on an inner side and to guides 2328 on anouter side. In turn, slide 2324 is connected through the Acme screw andpulley system to drive motor 2320, and the guides are connected throughthe lift platform to lifters 2308 a,b. The driver motor moves drive pins2352 through drive channels 2348 between a top position “A” closer totop edge 2340 and a bottom position “B” closer to bottom edge 2342. Thedrive pins are constrained to move horizontally, so that the pins pushagainst an interior side 2356 of drive channels 2348, urging cam unit2326 to move both horizontally and vertically. Guide pins 2354 insertedinto guidance channels 2350 are connected to relatively fixed portionsof the transport module, preventing horizontal motion, but permittingvertical motion, so that cam unit 2326 only moves vertically. As pin2352 moves between positions A and B, the pin moves a horizontaldistance H and a vertical distance V. It is the vertical displacementthat creates the raising and lowering motions. H and V may be optimizedfor particular plates and travel distances; in transport module 2100, Hand V are optimized for standard microplates and are approximately 10 cmand 3.5 cm, respectively. Cam unit 2326 is raised when drive pin 2352 isclose to position A, and cam unit 2326 is lowered when drive pin 2352 isclose to position B.

In use, a drive motor moves pins 2352 horizontally at a substantiallyuniform rate; consequently, the slope of drive channel 2348 determinesthe mechanical advantage and the rate of vertical motion. Close topositions A and B, the slope of drive channel 2348 is substantiallyzero, so that there is substantially no vertical motion. Stateddifferently, close to positions A and B, a preselected vertical positioncorresponds to a range of horizontal positions. This configuration makesthe vertical position relatively insensitive to motor precision ormanufacturing tolerance, because the lifter will be at the same verticalposition whenever it simply is near positions A or B. Between positionsA and B, the slope of drive channel 2348 is nonzero, so that there isvertical motion. The slope is largest (approximately 30°) betweenposition A and an intermediate position “C,” so that the lifter raisesand lowers relatively rapidly when it is farthest from the bottom of thestack of plates. The slope is smallest (approximately 15°) betweenpositions B and C, so that the lifter raises and lowers relativelyslowly when it is nearest to the bottom of the stack of plates.

The drive motor generally comprises any mechanism configured to generatea driving motion, as described above. The drive motor used in transportmodule 2100 is a stepper motor, which generates a constant torque.Generally, stepper motors and cams provide alternative mechanisms forperforming the same function, in this case, generating a varying rate ofmotion. However, pairing a stepper motor and cam together in theinvention provides several advantages. In particular, the cam providesmechanical advantage and positional insensitivity, and permits thestepper motor to be run at an optimal velocity profile. If the steppermotor were used alone, a much larger motor would be required to producethe required forces. Conversely, if the cam were used alone, with anonstepper motor, a system of limit switches would be required to limittravel and provide positional feedback to release the latches.

3. Intersite Drivers

FIG. 24 shows an intersite driver 2400, which generally comprises anymechanism for moving samples between different I/O and/or functionsites. Intersite driver 2400 includes a tray 2402 for supporting a plateand a shuttle mechanism 2404 for moving the tray (and an associatedplate) between I/O and/or function or transfer sites. The tray mayinclude a substantially planar platform 2406 to support the plate and arim-like plate guide 2408 that partially surrounds the platform toposition and partially secure the plate. The tray also may includeapertures (or recesses) 2410 for passage of a lifter (such as lifters2308 a,b in FIG. 23) from an intrasite driver for use in raising orlowering plates supported by the tray. The shuttle mechanism includes amotor 2412, a pulley system 2414, a drive belt 2416, and a guide shaft2418. The motor is connected to the drive belt through the intermediatepulley system. The tray is connected to the drive belt and slidinglymounted about the guide shaft.

In use, the motor turns the intermediate pulley system, which in turnmoves the drive belt, which in turn moves the tray, which moves along atrajectory specified by the guide shaft. Suitable motors include anymechanism for generating a force and/or torque, including but notlimited to a stepper motor. The tension in the drive belt may bemaintained using a tensioning spring attached to one end of the belt.

The transport module moves an input plate generally along an axis (e.g.,an x-axis) from an I/O site to the transfer site and moves an outputplate generally along the same axis but in the opposite direction fromthe transfer site to an I/O site. The input plate may be taken from thebottom of a stack of plates, and the output plate may be added to thebottom of the same or a different stack of plates, as described above.

4. Additional Features

The transport module may include additional features intended to enhancethe convenience and/or functionality of the module, such as processingbins and barcode readers, among others.

The I/O sites in the transport module may accommodate a variety ofcommercially available plates (e.g., microplates) and are large enoughso that the plates can be placed in the sites by a robot or a humanhand. Moreover, as shown in FIG. 3, the I/O sites also may accommodate avariety of commercially available pre- and postprocessing plate bins (ormagazines) for holding a stack of plates before and after analysis,respectively. A preprocessing bin may be removed from an I/O site andreplaced with another preprocessing bin containing a new stack of plateswith samples to be analyzed. Similarly, a postprocessing bin may beremoved from an I/O site and replaced with another postprocessing bin toreceive a new stack of plates with samples that have been analyzed. Theplate bins can be used with other robotics (such as an appropriatecombination of function modules) to dispense, wash, and read withoutrestacking plates. Preferred plate bins typically accommodate zero tosixty plates.

The transport module also may include barcode readers for automaticallyidentifying labeled plates. The barcode readers may be positioned ondifferent sides of the transfer site, so that the readers can readbarcodes mounted on different sides of a plate. The barcode readers alsomay be positioned to reduce specular reflection. The barcodes preferablyare read as plates are being raised, typically following transport ofthe plate from an I/O site to the direct transporter access site.Barcode readers may be selected to read at 700 scans per second orhigher and be programmed to decode a variety of symbologies, includingSPC (EAN, JAN, UPC), Code 39 (3-43 digits), Codabar (3-43 digits),Standard 2 of 5 (3-43 digits), Interleaved 2 of 5 (4-43 digits), Code 93(5digits), and MSI-Plessey (4-22 digits), among others. Informationobtained from the barcode can be used for various purposes. For example,the barcode can be used to convey instructions to the analyzer relatingto required changes in assay mode or optics configuration. The barcodealso can be used to name a report file.

5. EXAMPLES

The following examples illustrate the potential variety of singulationstrategies available in a transport module having the indicated numberof I/O sites and a capability for top and/or bottom input and/or output:

-   -   One I/O Site. The transport module may take plates from the        bottom or top of a stack of plates at a single I/O site and add        plates to the bottom or top of the same stack. There are four        possible singulation strategies:

BI/BO BI/TO TI/BO TI/TO

-   -    Here, B denotes bottom, T denotes top, I denotes input, and O        denotes output.    -   Two I/O Sites. The transport module may take plates from the        bottom or top of a stack of plates in either I/O site and add        plates to the bottom or top of the same stack in the same I/O        site and/or another stack in the other I/O site. There are        sixteen possible singulation strategies:

BI1/BO1 TI1/BO1 BI2/BO1 TI2/BO1 BI1/TO1 TI1/TO1 BI2/TO1 TI2/TO1 BI1/BO2TI1/BO2 BI2/BO2 TI2/BO2 BI1/TO2 TI1/TO2 BI2/TO2 TI2/TO2

-   -    Here, 1 denotes site 1, 2 denotes site 2, and B, T, I, and O        are defined as above. Significantly, if there are dedicated        input and output sites, the transport module may (1) take plates        from the bottom of the stack at the dedicated input site and add        plates to the bottom of the stack at the dedicated output        site, (2) take plates from the bottom of the stack at the        dedicated input site and add plates to the top of the stack at        the dedicated output site, (3) take plates from the top of the        stack at the dedicated input site and add plates to the bottom        of the stack at the dedicated output site, or (4) take plates        from the top of the stack at the dedicated input site and add        plates to the top of the stack at the dedicated output site.    -   Three or More I/O Sites. The transport module may take plates        from the bottom or top of a stack of plates at any I/O site and        add plates to the bottom or top of the same stack at the same        I/O site and/or another stack at one of the other I/O sites. For        a station with N I/O sites, there are generally (2N)² possible        singulation strategies.        The singulation strategy used by a particular transport module        may be fixed or varied from time to time or plate to plate. If        there are two or more I/O sites, a given site may be used for        input only, output only, or both input and output.

The I/O and function sites may be arranged to enhance convenience and/orefficiency, for both human users and the function modules. In transportmodule 2100, a first linear path connects the two I/O sites and thetransfer site, and a second substantially perpendicular linear pathconnects the transfer site and the dispense and examination sites.However, the I/O and function sites also may be arranged in other ways.For example, the I/O, dispense, and examination sites all may bepositioned along a single substantially linear path, which may betraversed in a single direction if plates are input at one end of thepath and output at the other end of the path.

If there are separate input and output sites (or separate input andoutput ends at a single I/O site), a robot may deliver a plate to theinput site and retrieve a (different) plate from the output site, bothin the same trip. This feature is termed “process compression,” becauseit reduces robot hand travel in servicing the transport module. Incontrast, if there is only a single input/output site (with delivery andretrieval from the same side), the robot would have to remove anyanalyzed plates before delivering any unanalyzed plates. Thus, processcompression replaces two separate robot movements with one robotmovement.

C. Fluidics Module

The fluidics module generally comprises any mechanism or system forautomatically dispensing fluid onto or into a sample holder. Themechanism may include reservoirs and dispense elements, and be capableof simultaneously and/or sequentially dispensing uniform and/ornonuniform fluid aliquots at one or more sample sites. The mechanismalso may include a thermal regulator to control fluid temperature,thereby reducing bubble formation. Further aspects of the fluidicsmodule are described in the following sections: (1) noncontact fluiddispensers, (2) contact fluid dispensers, (3) variable-pitch-array fluiddispensers, and (4) determination of inter-well separations.

1. Noncontact Dispensing

Fluid may be dispensed using “noncontact dispensing,” which generallycomprises any mechanism capable of dispensing fluid without contactingthe fluid and/or sample container into which the fluid is dispensed. Asimple example of a manual noncontact dispenser is an eyedropper, whichcan dispense drops of fluid without contacting a sample or receptacle.Further aspects of noncontact dispensing are described withoutlimitation in the following examples:

a. Example 1 Positive-Displacement Syringe Pump with Sapphire-TippedNozzles

FIG. 25 shows a noncontact fluid dispenser 3100 constructed inaccordance with aspects of the invention. Fluid dispenser 3100 mayinclude a fluid reservoir 3102, a pump 3104, and a dispense manifold3106 having at least one dispense element 3108.

Fluid reservoir 3102 generally comprises any container configured tohold fluid for dispensing by dispenser 3100. Fluid reservoir 3102 may beformed of any nonreactive material, including glass and variousplastics. Fluid reservoir 3102 may be configured so that it may beeasily replenished with fluid and/or easily replaced with an alternativefluid reservoir.

Pump 3104 generally comprises any device or mechanism configured to movefluid between fluid reservoir 3102 and fluid manifold 3106, and to meterfluid accurately to dispense elements 3108. In FIG. 25, pump 3104includes a (positive-displacement) microstepping syringe pump 3110incorporating a plurality of syringes 3112 for dispensing fluid to aplurality of dispense elements 3108. The syringe pumps may operateindependently using separate actuators, or the syringe pumps may beganged together and driven using a single actuator. Using one or moreaspirate/dispense valves 3113, syringe pump 3110 may aspirate fluidthrough aspirating tubes 3114 from fluid reservoir 3102, and dispensefluid through dispensing tubes 3116 toward dispense manifold 3106.Aspirating tubes 3114 and dispensing tubes 3116 may include anymechanism for transporting fluid between a fluid reservoir, pump, anddispense manifold, including but not limited to hollow plastic tubing.

Dispense manifold 3106 generally comprises any support configured tohold one or more dispense elements 3108. In FIG. 25, dispense manifold3106 is a substantially elongate bar that holds a linear array ofdispense elements, which may be used to dispense fluid into a lineararray of sample holders, such as rows or columns in a microplate. Eachdispense element can be driven by a separate pump, or two or moredispense elements can be coupled hydraulically using a manifold anddriven by the same pump.

Dispense element 3108 generally comprises any element configured todispense fluid to a surface, sample holder, or other repository,including surfaces and other fluids, without contacting such surface,sample holder, or other repository. In FIG. 25, dispense element 3108includes (1) a receiving portion 3118 configured to receive fluid fromdispensing tube 3116, (2) an anchor portion 3120 configured to connectthe dispense element and dispense manifold 3106, and (3) a dispensingportion 3122 configured to dispense fluid received through dispensingtube 3116. Receiving portion 3118 generally includes a connector forconnecting and securing together dispensing manifold 3106 and dispenseelement 3108. Anchor portion 3120 generally is joined to dispensemanifold 3106 using some suitable mechanism, such as passing the anchorportion through an aperture 3124 in the dispense manifold. Anchorportion 3120 may be formed of various materials, including substantiallyrigid materials such as steel tubing to stabilize and reduce recoil ofthe dispense element during dispensing.

Dispensing portion 3122 generally includes an exit port such as a nozzle3126 through which dispensed fluid may exit the dispense element. Theexit port may include a small cross-section orifice and/or a smalltapered tip and to increase the likelihood that dispensed fluid willexit the dispense element cleanly. The small cross-section orificeincreases the exit velocity of the fluid, enhancing the likelihood thatthe fluid has sufficient kinetic energy to overcome surface tension andinertia effects at low fluid volumes to separate cleanly from the tip.Alternatively, or in addition, the positive-displacement pump may beused to provide sufficient acceleration and exit velocity to the fluidto overcome surface tension, based on dispense volume, fluid viscosity,and other factors. The small and/or tapered tip reduces the surface areacapable of holding fluid, reducing the likelihood and potential size ofpendant drops. In FIG. 25, dispensing portion 3122 includes a200-micron-diameter sapphire orifice embedded in a dispense nozzle. Thedispense strategy also may include a suck-back feature to controlpendant drops and/or to increase predispense path length to allow ahigher exit velocity to be attained.

In an exemplary embodiment, the dispense element (3108) includes FEPtubing (3116) slid over a piece of metal tubing (3122) that has asapphire tip (3126) permanently embedded in an end. These elements aresurrounded by a machined tube with a large head (3120), which furthercompresses the FEP tubing and acts as a locational element in thedispense manifold (3106), setting the correct height and concentriclocation of the dispense element (3108). A screw-on fitting (3118)presses on the large head of the machined tube, pushing the dispenseelement (3108) into the tapped bore of the dispense manifold (3106),securing the dispense element in place.

Fluid dispenser 3100 may be used to dispense fluid to one or more sampleholders. Generally, a sample holder may be positioned beneath eachdispense element, and pump 3104 may then be used to dispense a meteredamount of fluid through each dispense element to each sample holder.Sample holders may be identified and sample holders and dispenseelements may be aligned using any suitable mechanism. Each portion ofthis operation may be under computer control, including alignment of thedispense elements and sample holders, and operation of the pump, amongothers.

Fluid dispenser 3100 may be capable of dispensing fluids over a widerange of fluid volumes, and particularly in a preferred range betweenabout 0.1 μL and about 100 μL. In assays, the coefficients of variation(CVs) for such dispenses preferably are about 2-10% or less fordispensed volumes down to about 0.1 μL. Fluid exit velocity may beoptimized for the dispensed volume, for example, to reduce splashing asthe fluid contacts the sample holder. The upper limit on dispense volumeis essentially unconstrained, except by pump capacity.

b. Example 2 Positive-Displacement Syringe Pump with Solenoid Valve andSapphire-Tipped Nozzles

FIG. 26 shows an alternative noncontact fluid dispenser 3150 constructedin accordance with aspects of the invention. Fluid dispenser 3150generally includes a fluid reservoir 3152, a pump 3154, and a dispensemanifold 3156 having at least one dispense element 3158. Thesecomponents function essentially as described above for fluid reservoir3102, pump 3104, dispense manifold 3106, and dispense element 3108,respectively.

Fluid dispenser 3150 also includes a pulse element 3160, which may beused to create a pressure wave as the pulse element is opened and closedto increase the energy of the fluid being dispensed. Pulse element 3160preferably comprises a solenoid valve mounted near the dispense element,and coupled to the dispense element via an inlet fitting 3162. The pulseelement may be pulsed or left open during the dispense. Each dispenseelement may be associated with its own pulse element, or groups ofdispense elements may share a pulse element. Each pulse element may beunder computer control.

c. Example 3 Positive-Pressure Pump with Solenoid Valve andSapphire-Tipped Nozzles

FIG. 27 shows another alternative noncontact fluid dispenser 3200constructed in accordance with aspects of the invention. Fluid dispenser3200 may include a pressure-responsive fluid reservoir 3202, a pressurepump 3204, and a dispense manifold 3206 having at least one dispenseelement 3208. Fluid may be dispensed from fluid reservoir 3202 todispense elements 3208 using one or more dispense tubes 3209.

Pressure-sensitive fluid reservoir 3202 generally comprises anycontainer configured to hold a fluid for dispensing, and to respond toan increase in pressure by dispensing a fluid. In FIG. 27,pressure-sensitive fluid reservoir 3202 comprises a fluid bladder, whichresponds to external pressure by decreasing in volume, concomitantlyextruding or dispensing fluid.

Pressure pump 3204 generally comprises any device or mechanismconfigured to apply a variable but controllable positive pressure topressure-sensitive fluid reservoir 3202. Pressure pump 3204 operates bycompressing pressure-sensitive fluid reservoir 3202, forcing fluid fromthe reservoir through dispense tube(s) 3209 to dispense elements 3208.In FIG. 27, pressure pump 3204 includes a pressure source 3210, anupstream pressure controller 3212, a pressure vessel 3214, and adownstream pressure controller 3216. Pressure source 3210 may includeany source of positive pressure, including a pump and/or a pressuretank, among others. Upstream pressure controller 3212 may include anymechanism for monitoring and/or regulating the pressure produced by thepressure source, and may be under manual or automatic control. Pressurevessel 3214 may include any container capable of enclosingpressure-sensitive fluid reservoir 3202 and maintaining a positivepressure on such reservoir. Downstream pressure controller 3216 mayinclude any mechanism for monitoring and/or regulating the pressure offluid in dispense tube(s) 3209.

Positive-pressure pumping preferably should be performed so thatadditional gases cannot go into solution in fluids being dispensed,because this could cause bubbles as the pressure is decreased duringdispensing. Such additional gases may be avoided as shown above by usingpreviously degassed fluids in a flexible bladder that is pressuredexternally in a pressure vessel.

Dispense manifold 3206 and dispense elements 3208 may take variousforms, including forms described above for fluid dispensers 3100 and3150. In FIG. 27, a single dispensing tube 3209 is used to supply fluidto the dispense elements 3208, and the dispense elements include pulseelements 3218, as described above. The pulse element is used to meterthe fluid by timing the duration of the valve opening. This requires thefluid to have a controlled and calibrated flow rate. The pulse elementalso aids fluid dispensing, as described above, because the pressurewave aids fluid separation from the dispense element.

Alternative fluid dispensers may use alternative combinations ofcomponents. There may be a single pressure source for each dispenseelement, or there may be a common pressure source for sets of dispenseelements. There may be a single pulse element for each dispenseelements, or there may be a common pulse element for sets of dispenseelements. The pressure drop in the fluid after the pulse element foreach fluid path must be approximately the same for a common pulseelement. Single pulse elements for each dispense element may haveindividually calibrated open times or duty cycles during the open timeto account for differences in pressure drop.

d. Example 4 Positive-Displacement Syringe Pump with a Rectangular Arrayof PTFE Nozzles

FIG. 28 shows yet another alternative noncontact fluid dispenser 3300constructed in accordance with aspects of the invention. FIG. 28 is aschematic view of the fluid dispenser shown (together with a transportmodule and an analysis module) in FIG. 2 as part of an integrated systemfor preparing and/or analyzing samples. Fluid dispenser 3300 may includea fluid reservoir station 3302, at least one pump 3304, and at least onenoncontact dispense elements 3306. These components may functionessentially as described above in the context of FIGS. 25, 26, and/or27. In use, the pumps direct fluid from the fluid reservoir stationthrough the dispense elements and onto or into a sample holder such as amicroplate 3308.

Fluid reservoir station 3302 may include bottles or fluid containers3310 for holding buffers, reagents, samples, or other fluids for use ina particular assay. Fluid containers 3310 may vary in size, depending onfluid requirements for a particular procedure. For example, a largecontainer may be used to dispense a buffer used in many sequentiallyperformed assays. Alternatively, a small container may be used todispense a tracer used in relatively small quantities and/or in arelatively small number of assays. Fluid reservoir station 3302 may beconfigured to facilitate easy interchange of different fluid containersfor different purposes, for example, by using containers having asnap-on or screw-on connector. Fluid containers 3310 may be disposableor reusable and may be supplied independently or with a particular assaykit. The number of reservoirs that may be used simultaneously in station3302 may range from 1 to N, where N is the number of dispense elementassemblies that are connected to station 3302.

The dispenser effectively comprises an interchangeable conduit networkthat allows any combination of dispense elements (or tip devices) to beconnected to any combination of fluid reservoirs. Dispenser 3300 mayinclude one or more pumps 3304 such as syringe pumps and one or moredispense elements 3306. Generally, any pump may be connected to anyfluid container, for example, via an aspiration tube 3312. Thus,multiple pumps may be connected to one or some fluid containers, and nopumps connected to other fluid containers. Alternatively, each pump maybe connected to a different fluid container. Similarly, any pump may beconnected to any dispense element, for example, via a dispense tube3314. Thus, one pump may be connected to one or multiple dispenseelements, and so on. In a preferred configuration, the dispenserincludes 32 separate syringe pumps 3304 each connected one-on-one viaseparate dispense tubes 3314 to 32 separate dispense elements 3306.

The syringe pump may include various drivers. For example, the syringepump may include a linear stepper motor or a linear servo motor.Alternatively, the syringe pump may include a rotary stepper motor or arotary servo motor.

Dispense elements 3306 generally may be organized in any suitablearrangement, including linear and rectangular arrays. In mostapplications, it is preferable to match the organization of the dispenseelements to the organization of all or part of the sample sites in thesample holder. Here, sample sites are preferred sample locations withinthe sample holder. Thus, a periodic (e.g., rectangular, hexagonal, etc.)arrangement of dispense elements is preferable for a sample holder suchas a microplate or biochip having a periodic array of sample sites. Inessence, the dispense elements are arranged to form an array of fluiddispensing channels that correspond to the sample sites. In a preferredconfiguration, the dispenser includes 32 dispense elements organized asa 4×8 regular array 3316, and more specifically as a 4×8 rectangulararray with 9 mm separations to correspond to the 9 mm well separation instandard 96-well microplates, as described above.

Dispense-element array 3316 may be used to dispense individual measuredaliquots of fluid onto or into a sample holder. For example, the arraymay be used to dispense into some or all of the wells 3318 in microplate3308 by aligning the array with the wells and dispensing. If there aremore sample wells than dispense elements, dispensing can occur in stepsby dispensing to a first set of wells 3322, moving the dispense arrayand microplate relative to one another (for example, along a Y-axis),and then dispensing to a second set of wells 3324. This process may berepeated for additional sets of wells 3326 as necessary. This process isillustrated in FIG. 28, in which three sets of dispenses from a 4×8array are used to dispense into a 96-well microplate.

A similar process may be used to dispense fluid into a microplate thathas a higher density of wells, for example, 384-wells, or 1536-wells,among others. This can be accomplished by offsetting the dispenseelement array 3316 and/or microplate 3308 in the X direction and doingnumerous passes in the Y direction. For example, two full passes in theY direction with one adjustment in the X direction will allow dispensingin each well of a 384-well microplate. Similarly, four full passes inthe Y direction with three adjustments in the X direction will allowdispensing in each well of a 1536-well microplate. The adjustment oroffset should be by an integer multiple of the well-to-well spacing.Dispensing by column into 96, 384, 864, 1536, and 3456 well microplatescan be accomplished using a linear array of 8 dispensing tips, since thenumber of rows is 8, 16, 24, 32, and 48, respectively. Dispensing by rowinto 96, 384, 864, 1536, and 3456-well microplates can be accomplishedusing a linear array of 12 dispensing tips since the number of columnsis 12, 24, 36, 48, and 72 respectively. Any microplate with a number ofcolumns or rows that is divisible by 8 can be dispensed into with thismethod. A rectangular array of dispensing tips may also be used with thecenter-to-center spacing of 9 mm in both directions, since thewell-to-well spacing in all the above-mentioned plates is 9 mm or aninteger fraction thereof (e.g., 4.5 mm, 2.25 mm, etc.).

FIG. 29 shows a portion of fluid dispenser 3300 including a dispensedriver 3400 and an array 3402 of dispense elements 3404 positioned abovea microplate 3406 at a dispense site. (The distance between the dispensedriver and microplate has been exaggerated for clarity.) FIG. 29 is apartial view of fluid dispenser components shown in FIGS. 2, 3, and 18relative to transport and/or analysis modules. The fluid dispensergenerally may include (or associate) registration mechanisms for movingthe dispense array and/or the microplate or other sample holder relativeto one another along X, Y, and/or Z axes for dispensing. Such movementmay be effected using any suitable combination(s) of drive mechanismsand any suitable drive strategy. In fluid dispenser 3300, the microplateis moved relative to the dispense array along the X and Y axes, and thedispense array is moved relative to the microplate along the Z-axis.More specifically, the microplate is moved using a transporter sharedwith an analysis module, as described below in connection with theanalysis module, and the dispense array is moved using dispense driver3400.

Dispense driver 3400 includes a linear actuator 3410 and parallel slides3412 a,b directed along the Z-axis on opposite sides of array 3402. Theactuator moves the dispense array along the Z-axis guided by theparallel slides, so that the array may be raised and lowered relative tothe microplate. Here, the linear actuator includes a stepper motor 3414and an Acme screw 3416, although any mechanism capable of generating alinear displacement may be used, as described above in connection withthe intrasite driver used in the fluidics module.

The array of dispense elements generally may be formed from one or morebanks 3420 of dispense elements. In fluid dispenser 3300, these bankseach include 8 dispense elements, which may be driven directly by apositive displacement pump or by a manifold. The banks may be positionednear one another with a spacing corresponding to the spacing betweeninteger numbers of sample sites, for example, about 9 mm for standardmicroplates. This spacing may reduce spatial requirements whilepreserving an ability to dispense into the entire microplate. Thisspacing also may permit dispensing of multiple reagents with one scan ofthe sample holder under the dispenser array.

Each bank of dispensers can be independently installed or de-installedinto a standard slot arrangement. With this slot arrangement, banks ofdispense tips with different dispense characteristics (e.g., number oftips, volume range, or other functions such as plate washing) may beinstalled in a mix and match fashion. Software can be configured for thetype of module that has been installed into each slot, and programmedaccordingly. For example, microplate washing can be implemented bychanging the design and programming of each bank of dispense elements.With proper design and sizing, one bank of dispense elements canaspirate solution from a column or row of wells, while another bank cansubsequently dispense clean solution. Alternately, for the washfunction, a head may contain both dispense and aspirate elements, atdifferent heights, allowing dispense and aspirate without movement ofthe plate.

When each dispense element is connected to a separate pump, a softwareprogram can control the pumps while the plate is being scanned to allowrandom access dispensing of any reagent into any well. In paralleldispensing or transfer systems (e.g., pin transfer devices or arrays ofconventional pipettes), random access is only possible if a source plateis first created with the desired reagent distribution or if thereagents somehow can be routed into the tops of the pipettes fromseparate sources. The dispenses from each dispense element may beperformed simultaneously or sequentially, and be of uniform ornonuniform aliquots.

FIG. 30 shows a fluid control unit 3400 for use with the fluid dispenserof FIG. 28; alternative views/embodiments are shown in FIGS. 2 and 3 inrelation to optionally associated transport and analysis modules. Thefluid control unit generally comprises the pump or pumps and associateddrivers used to direct fluid from a fluid reservoir station to one ormore dispense elements. Fluid control unit 3400 includes a plurality ofpumps 3402 (such as syringe pumps) and associated drivers 3404 mountedin a chassis 3406. The syringe pumps include a syringe 3410 and aninlet/outlet valve 3412. The syringe is used to aspirate and dispensefluid and includes a barrel 3414 for holding fluid and a plunger 3416slidably received within the barrel and capable of generating a positivedisplacement. The plunger may be operatively connected to driver 3404.The valve is used for fluid input/output and includes an inlet 3418 forconnecting to an aspiration or input tube 3419 coming from a fluidreservoir and an outlet 3420 for connecting to a dispense or output tube3421 going to one or more dispense elements. Fluid reservoirs may beplaced adjacent the fluid control unit to provide convenience inoperation.

The valve or associated pump may include labels 3422 to assist hookup ofinput and output tubes. For example, valves and/or pumps associated witha control unit for a 4×8 array of dispense elements may be labeledA1-H1, A2-H2, A3-H3, and A4-H4, where the A-H are the standarddesignators for the 8 rows in a standard 96-well microplate, and the 1-4refer to four columns. Moreover, valves, pumps, and/or input/outputtubes, or portions thereof, may be color-coded, for example, using red,yellow, blue, and green to denote 1-4, as defined above. Generally, anymarking or component capable of distinguishing valves, pumps, and/ortubes may functions as markings; however, preferred markings are numberand/or letter codes and colors.

A preferred syringe pump is a CAVRO syringe pump. The starting speed ofthe CAVRO syringe pump is 1000 ½ steps/second, and the associatedacceleration (slope) is 50,000 ½ steps/second². The pump executes 6000 ½steps in its 30-mm travel, so that its starting speed is 5 mm/second,and its associated acceleration is 250 mm/second². If used with a 500-μLsyringe, there are about 12½ steps per μL, so that the starting speedand top speed are not too different with the relatively small volumes(e.g., 0.5 μL) used here.

FIG. 31 shows a bank of dispense elements 3500 for use with the fluiddispenser of FIG. 28. The bank includes a manifold 3502 and a pluralityof substantially equally spaced dispense elements 3504 each attached toa tubing assembly 3506. The manifold supports the dispense elements andmay be used to affix the bank to a dispense driver using suitableaffixing means, such as fasteners 3508. The manifold may be formed ofany suitable material, such as stainless steel, which is resistant tofluids and other materials that may come into contact with the manifold.The manifold may be asymmetric to ensure that it is mounted properly ata dispense site. The tubing assembly is used to connect the dispenseelements to the fluid reservoir(s). The manifold and/or tubingassemblies may include labels 3510 a,b,c to assist hookup between themanifold and assemblies.

FIGS. 32 and 33 show alternative views of bank 3500 and associatedtubing assemblies 3506. The tubing assembly may include an input(distal) flange 3520 for connecting to a pump (e.g., in a fluid controlunit), an output (proximal) flange 3522 for connecting to a dispenseelement, and a section of tubing 3524 for spanning the gap between thepump and the dispense element. The input and output flanges generallycomprise any suitable mechanism for ending the tubing so that it may bejoined to another structure. For example, the flanges may include ¼-28fittings. The tubing generally may have any suitable dimensions. Forexample, the tube may be about 40 inches long to accommodate positioningof the pumps relative to the dispense array, with an inner diameter ofabout {fraction (30/1000)} of an inch and an outer diameter of about{fraction (62/1000)} ({fraction (1/16)}) of an inch. The tubing furthermay include reinforcements such as a spiral wrap strain relief 3526positioned adjacent the output flange. A preferred tube material is FEP.The dispense element may include a head (or tip flange) 3530, a bodytube 3532, and a dispense tip 3534. The tubing assembly and anassociated dispense element may be operatively joined by inserting bothinto an appropriately sized aperture 3540 in manifold 3502, separated bywashers 3542 a,b and an intervening O-ring 3544.

The fluid enters the dispense element at the interface of the tip flangeand supply tubing flange. This flanged interface may reduce fluid holdupand dead volume areas, important when changing fluids or cleaning thetip, and may allow for either the tip or supply tubing to be changedindependently. The tip flange may be formed via a machined PEEK plasticsection of the tip. The tubing flange may be formed conventionally. Thismachined flange is press-fit onto a stainless steel tube. The entireinner surface of the PEEK flange and stainless steel tube is lined withthin-wall PTFE heat-shrink tubing, terminating at the PEEK flange at oneend and the dispense nozzle 3550 at the other (dispense) end. Thedispense end is formed from the heat-shrink tubing into a cone-shapednozzle protruding from the stainless steel tubing. The thin wall of theheat-shrink tubing and its ability to shrink onto a mandrel areimportant features of the dispense element. The cone-shaped nozzle maybe formed manually. This process should be performed carefully andreproducibly, since a burr on the dispense end, an unsquare cut of thedispense end, or a slightly misshaped nozzle all may affect dispensingperformance. Alternatively, the dispense element may include aconventionally injected molded tip made of polypropylene withsubstantially similar geometry. The wall thickness and aperture openingwould be of similar size, and care would have to be taken to ensure thatno flash from the molding process affected dispensing performance.

The noncontact flanged dispense tip described here shares many featureswith the sapphire dispense tip described above, including a small,controlled, inner-diameter bore and a small-to-minimum surface area atthe dispense end of the tip. The inner diameter of the orifice of thenoncontact flanged dispense tip is about 190±10 microns, and the innerdiameter of the orifice in the sapphire tip is about 200 microns. Thecircumferential wall of the tip around the dispense orifice is about5-thousandths of an inch thick. The small bore increases the exitvelocity of the fluid, and the minimum surface area decreases surfacetension, both of which are important for cleanly ejecting small (˜0.5μL) drops of fluid. The noncontact flanged dispense tip also provides acompletely nonmetallic flow path because the interior of the flow pathis PTFE Teflon. This greatly reduces the formation of pendant drops dueto the surface properties of the Teflon, which is an improvement overthe design of the sapphire tip. Generally, any material (such as ahydrophobic material) that reduces the affinity of the dispensed fluidfor the dispense element may be used within the fluid path and/or at thedispense tip to reduce pendant drops. Such materials includepolypropylene, polyethylene, and FEP.

The system described above may be used to dispense fluid volumes down to0.25 μL or less. Small (submicron) volumes generally may be dispensed bydecreasing the thickness of the wall of the tip, especially to or belowabout 8-thousandths of an inch, and even to or below about2.5-thousandths of an inch. Generally, a wall thickness of about10-thousandths of an inch does not work well for dispensed volumes ofless than about 1 μL. Smaller dispensed volumes also may be obtained byusing a syringe pump with a linear motor, which may also increase therate of fluid dispensing by providing a more rapid acceleration and thusa higher exit velocity.

In summary, the noncontact dispenser may include a fluid source, a pump,and a noncontact dispenser, where a conduit path extends from the pumpto the orifice of the dispenser. The conduit path may remain open andunconstricted between successive depositions because fluid is retainedin the conduit path by surface tension and/or capillary action untilexpelled by the positive displacement pump. Thus, the dispenser maydispense fluid without closing or constricting the conduit channel, orcontacting droplets of the dispensed fluid to a surface. Moreover, therate of deposition will generally correspond to or equal the incrementalrate of pumping. The dispenser may be used to deposit fluid aliquots assmall as 5 μL or less.

2. Contact Dispensing

Fluid also may be dispensed by “contact dispensing,” which generallycomprises any mechanism for dispensing fluid in which the dispensercontacts the sample and/or sample container into which the fluid isdispensed. An example of a contact dispenser is a pin transfer device,which uses a pin to pick up small quantities of fluid from a storagearea and transfer the fluid to a receptacle. Pin transfer devices aredescribed below in the context of microplates, although they also may beused for transfer to and/or from other sample containers. Furtheraspects of contact dispensing are described without limitation in thefollowing examples:

a. Example 1

FIG. 34 shows a pin transfer device 3700. Pin transfer device 3700includes a pin 3702 and a mount 3703 configured to support the pin sothat a tip 3704 of the pin is presented for fluid transfer. Pin transferdevice 3700 may be used to transfer a small amount of a first liquid3706 from a storage area 3708 to a second liquid 3710 in a receptacle3712. This transfer may proceed in two steps:

-   -   Step 1. For loading, pin transfer device 3700 is positioned over        storage area 3708 (Panel A), lowered until tip 3704 contacts        first liquid 3706 (Panel B), and then raised until tip 3704        breaks contact with first liquid 3706 (Panel C). In the process,        a drop 3714 of first liquid 3706 remains in contact with tip        3704 due to surface tension.    -   Step 2. For dispensing, pin transfer device 3700 is positioned        over receptacle 3712 (Panel D), lowered until tip 3704 and drop        3714 contact second liquid 3710 (Panel E), and then raised until        tip 3704 breaks contact with second liquid 3710 (Panel F). In        the process, drop 3714 will be transferred to second liquid        3710. (A new drop 3716 representing a mix of second liquid 3710        and drop 3714 may remain in contact with tip 3704 after the        transfer.).

The volume of drop 3714 will depend on various factors, including (1)the surface tension and viscosity of first liquid 3706, (2) thematerial, geometry, and cleanliness of tip 3704, and (3) the kinetics oftransfer. However, for a fixed set of parameters (e.g., fluid, tip,etc.), the volume should be relatively precise and reproducible,permitting volumetric fluid transfer.

b. Example 2

FIG. 35 shows an alternative pin transfer device 3750. Pin transferdevice 3750 includes a plurality of pins 3752 and a rigid mount 3754configured to support the pins in a preselected array. The tips 3756 ofpins 3752 lie approximately within a plane 3758. Pin transfer device3750 is configured to transfer a drop of fluid 3760 simultaneouslybetween arrays of storage areas and/or receptacles, such as wells 3762in a microplate 3764. More specifically, the device is configured totransfer fluid substantially simultaneously between storage areas andreceptacles by substantially simultaneously contacting the pins to thestorage area(s) to load the fluid, and then substantially simultaneouslycontacting the loaded pins to the receptacle(s) to unload the fluid.

Unfortunately, pin transfer devices using a rigid array of pins maysuffer from a number of shortcomings. For example, there may bevariations in the dispensed volume if the receptacles do not lie in asingle plane, because the tips of some of the pins may not be able tocontact the associated receptacle. There also may be variations in thedispensed volume if the receptacles are unevenly spaced perpendicular tothe plane of the tips, because some of the pins may miss the associatedreceptacle. These shortcomings may be particularly acute for transfer toand/or from microplates, because microplates may vary in theirdimensions due to shrinkage or expansion during or after molding andbecause microplate wells may have uneven bottom surfaces.

c. Example 3

FIGS. 36 and 37 show another alternative pin transfer device 3800configured to transfer fluid simultaneously between arrays of storageareas and/or receptacles. Pin transfer device 3800 includes a pluralityof pins 3802 and a flexible mount 3804 configured to support the pinssecurely but movably in a preselected array. The flexible mount mayallow the pins to move vertically and/or horizontally relative to theirequilibrium positions. In pin transfer device 3800, flexible mount 3804includes a rigid block 3806, a film 3808 abutting the rigid block, aflexible (or elastomeric) sheet 3810 abutting the film, and a holder3812 abutting and at least partially holding the block, film, and sheet.Pins 3802 are embedded in flexible sheet 3810 and slidably positionedthrough apertures 3814 in film 3808 and rigid block 3806. Portions ofthe pins configured to contact the flexible sheet may be etched tofacilitate adhesion with the sheet. Rigid block 3806 provides structuralrigidity that maintains the orientation and relative positions of thepins. Film 3808 separates rigid block 3806 and flexible sheet 3810,permitting the flexible sheet to move independently of the rigid block.Flexible sheet 3810 provides a force that biases pins 3802 toward rigidblock 3806. Flexible sheet 3810 may include one or more layers,providing a flexible and chemical-resistant surface and the desiredcompliance and mechanical stability. Film 3808 and/or flexible sheet3810 may provide a barrier to fluid flow through the device. Pintransfer device 3800 may be used to transfer fluid to a microplate 3820having a plurality of wells 3822.

A flexible mounting mechanism may overcome shortcomings associated withrigid mounts. For example, a flexible mechanism may allow pins to moverather than bend or break if accidentally brought into contact with asurface such as a shallow well bottom during fluid transfer. A flexiblemechanism also may be used to compensate for variations in sample-holderdimensions, such as variations due to shrinkage and/or expansionrelative to the “nominal” dimensions. For example, the flexible sheetmay be mounted in a frame whose outer dimensions can be adjusted to beslightly larger or slightly smaller than the nominal dimensions, forexample, by placing the sheet under stress. The pin-to-pin spacing canthen be reduced to match a smaller sample holder by decreasing thestress on the sheet (e.g., by adjusting the outer frame), and increasedto match a larger sample holder by increasing the stress on the sheet.

The flexible mounting mechanism in pin transfer device 3800 may overcomeshortcomings associated with other flexible mounts, such as flexiblemounts that simply permit pins to float in their mounts. For example,the flexible sheet may inhibit the accumulation of solids on the pinsnear the anchor points of the pins, reducing friction between the pinsand their fixtures. Such friction may prevent the pins from movingfreely or at all, so that some pins may not be able to touch the desiredsurface and transfer fluid uniformly. The sheet also may facilitatecleaning, because the ends of the pins are sealed in the sheet, reducingcontamination by cleaning materials. Typically, the device is cleaned byimmersing and/or scrubbing portions of the device that contact fluid,such as the tips of the pins, with a cleaning fluid.

Pin transfer device 3800 generally may be formed or constructed usingany suitable technique, including the following five-step process.First, pins 3802 may be positioned in a rack. Second, pins 3802 may bepositioned through rigid block 3806 and film 3808. Third, flexible sheet3810 may be poured to encapsulate the pins, and allowed to dry. Fourth,holder 3812 may be mounted to the sandwich formed by the rigid block,film, and flexible sheet. Finally, the finished device may be removedfrom the rack.

Pin transfer device 3800 also generally may be formed of any materialshaving the desired mechanical properties. For example, the flexiblesheet may be formed of any suitable flexible material, and the pins maybe formed of any suitable wetable material that facilitates the loadingand unloading of reproducible volumes of fluids. Preferred materialsinclude a silicone RTV sheet and a closed cell polyurethane foam for themount and stainless steel for the pin.

The pin transfer device may be used manually and/or automatically. Forexample, pin transfer device 3800 includes apertures 3830 for mountingthe device to a driver for raising and lowering the device relative to asample, and/or for moving the device between different samples.

The pin transfer device also may be used in conjunction with anysuitable sample holder, including microplates. If used with amicroplate, the pin transfer device may include linear or rectangulararrays of pins corresponding to some or all of the rows or columns in amicroplate having 96, 384, 864, 1536, 3872, 9600, or another number ofwells. In turn, the microplate or other sample holder may be constructedto transfer information regarding the form of the microplate to the pintransfer device. Such information may include the dimensions of thesample holder and the number and dimensions of any associated samplewells. Such information may be encoded using a bar code, a referencefiducial, and/or other features of the sample holder.

3. Variable Pitch Array Fluid Dispenser

The separations between dispense elements in a fluid dispenser may befixed to correspond to nominal separations between sample sites instandard sample holders, or integer multiples thereof, as describedabove. However, if the separations between sample sites in actual sampleholders differ significantly from the nominal separations in thestandard sample holders, these fluid dispensers may dispense fluid ontothe side or outside of the intended sample sites in the sample holder.Such misdirected dispenses may be especially likely with microplatesample holders, because microplate dimensions often vary due toshrinkage and/or expansion during or after molding. To overcome thesedifficulties, the fluid dispenser may include mechanisms for actuallyand/or effectively adjusting the separation between dispense elements tomatch the separation between sample sites in a given sample holder.

FIGS. 38 and 39 show a variable-pitch-array fluid dispenser 3900 thatmay be used effectively to adjust the separation between dispenseelements. Fluid dispenser 3900 includes a dispense manifold 3902 and adispense site 3904 configured to receive a sample holder 3906 having aplurality of sample wells 3908.

Dispense manifold 3902 may take various forms. For example, dispensemanifold 3902 includes a substantially linear array of dispense elements3910. The manifold includes a fluid inlet 3912 positioned adjacent afirst end 3914 of the array and a pivot 3916 positioned adjacent asecond end 3918 of the array. Fluid inlet 3912 is operatively connectedto dispense elements 3910, so that fluid may enter dispense manifold3902 through fluid inlet 3912 and exit dispense manifold 3902 throughdispense tips 3920 of dispense elements 3904. Fluid 3922 may bedispensed through dispense tips 3920 using a variety of mechanisms,including noncontact mechanisms, as described above. The fluid may exitthe dispense tip in various forms, including drops, trains, and streams,among others, depending on exit velocity, dispense duration, orificediameter, and so on.

Dispense manifold 3902 is rotatable about pivot 3916, so that theeffective separation between dispense elements 3910 can be adjusted tomatch the actual separation between sample wells 3908 in sample holder3906. Specifically, dispense manifold 3902 may be rotated relative to asample holder, so that the array of dispense elements is oriented at anangle θ relative to the array of sample wells. Rotation of the array ofdispense elements relative to the sample holder effectively decreasesthe separations between dispense elements relative to the sample holder.Mathematically, the effective separation between linearly arrayeddispense elements is described by the following equation:S _(eff) =S·cos θ  (1)Here, S_(eff) is the effective separation between dispense elements, Sis the actual separation between dispense elements, and θ is the anglebetween the array of dispense elements and the array of sample wells.Generally, the actual separation between dispense elements in thedispense manifold should equal or exceed the maximum separation betweensample wells in expected sample holders, because relative rotation ofthe dispense manifold and sample wells can only decrease the effectiveseparation between the dispense elements.

Rotation of the dispense manifold about the pivot generally may becontrolled automatically by the dispenser or manually by an operator. Ifthe pivot is controlled automatically, a driver may be connected to thepivot or to the dispense manifold, depending on whether the dispensemanifold is attached fixedly or rotatably to the pivot, respectively.

Fluid is dispensed into a sample holder by adjusting the relativepositions of the dispense elements and sample holder so that S_(eff) issubstantially equal to the actual separation between sample sites, andthen by moving the sample holder relative to the dispense site whilesimultaneously or sequentially dispensing fluid from the dispense tipsof the dispense elements. Fluid may be dispensed simultaneously if theangle of rotation θ is small, so that each dispense element ispositioned over a sample well simultaneously. Fluid may be dispensedsequentially if the angle of rotation θ is large, so that only somedispense elements are positioned over a sample well at a given time. Inthe latter case, fluid dispensing must be coordinated with the relativemotion of the sample holder. If the sample holder includes multiple rowsof sample wells, fluid may be dispensed sequentially into each rowthrough cycles of motion and dispensing, the cycles of relative motionbringing subsequent rows of sample wells into alignment with thedispense manifold. Generally, the sample holder may be moved, thedispense manifold may be moved, or both may be moved for fluiddispensing.

The relative positions of the sample holder and dispense site may bealtered using any suitable mechanism. For example, the dispense manifoldmay be rotated and/or translated relative to the sample holder, thesample holder may be rotated and/or translated relative to the dispensemanifold, or the sample holder and dispense manifold may be rotatedand/or translated relative to one another. Similarly, the dispensemanifold and/or the sample holder may be rotated about a variety ofpositions, so that the pivot may be located at various positions withinthe dispense manifold and/or dispense site.

Alternative mechanisms also may be used to adjust the separation betweendispense elements in a fluid dispenser to correspond to the separationof sample wells in a sample holder. In one alternative, the separationbetween each pair of dispense elements may be adjusted to correct fordeviations in positions of sample wells along a first axis, and therelative distance moved by the fluid dispenser and sample holder may beadjusted to correct for deviations in positions of sample wells along asecond axis. In another alternative, if each dispense element can beindividually controlled, a linear array of dispense elements and samplewells may be moved parallel to one another, coordinating the dispensingof fluid with the motion. This requires closely coordinating dispensingand motion, and is essentially a group of single dispenses. In yetanother alternative, each linear array in a rectangular array ofdispense elements may slide relative to one another, so that elements ineach linear array may rotate and translate to establish and maintainalignment with rows of sample wells.

The variable-pitch array fluid dispenser generally may be used withvarious sample holders and various types of dispensing. For example, thedispenser may be used with microplates and surfaces. Similarly, althoughthe dispenser was described primarily in the context of noncontact fluiddispensing, aspects of the invention could be used to position a contact(e.g., pin transfer) fluid dispenser relative to sample wells in asample holder.

4. Determination of Inter-Well Separations

The fluid dispenser also may include mechanisms for determining theseparation between sample positions in a sample holder, and forcommunicating these separations to an operator and/or an automatedcontroller. These mechanisms may be used to position a sample holderrelative to a fluid dispenser, and/or to position a sample holderrelative to other devices, such as excitation and/or emission elementsin a light detection device. FIGS. 38-39 show several of thesemechanisms.

Sample positions may be determined using any mechanism capable ofmeasuring positions, either before or during fluid dispensing.Information regarding sample positions may be determined before fluiddispensing by inspecting individual sample holders, or by inspectingrepresentative sample holders in a batch of sample holders. The latterapproach would be most successful if plate-to-plate variations withinthe batch are small. Information regarding sample positions may beprovided to a controller for the dispenser or other device, eithermanually or automatically. For example, information could be enteredmanually by an operator, as at a keyboard. Alternatively, informationcould be entered automatically by encoding the information on the sampleholder and then reading the information prior to dispensing. Forexample, information could be entered using a bar code 3930 on thesample holder and a bar code reader associated with the dispenser.

Information regarding sample positions also may be determinedimmediately before or during fluid dispensing by inspecting individualsample holders. One method for determining sample positions is to use animaging device 3932, such as a camera, and image recognition software toidentify the actual location of sample positions. Another method is touse a light detection or other device to locate reference fiducials 3234associated with the sample holder, and to identify the positions ofsample wells from the positions of reference fiducials by interpolationand/or extrapolation. Such reference fiducials could include dedicatedfeatures, and/or specific sample positions or edges of the sampleholder, among others, as described in PCT Patent Application Ser. No.PCT/US99/08410, filed Apr. 16, 1999, and incorporated herein byreference.

Information regarding positions of sample wells may be used by variousfunction modules, including a fluidics module, as here, or an analysismodule, as described below.

D. Auxiliary Modules

This section describes auxiliary function modules that may be used aloneas stand-alone units or together with or in lieu of fluidics and/oranalysis modules in an integrated system for sample preparation and/oranalysis. An auxiliary module generally comprises any mechanism orsystem for performing functions that complement or assist fluiddispensing and/or analysis. Exemplary auxiliary modules include amongothers (1) a cleaning module, (2) a sample-containment module, and (3)an incubation module.

1. Cleaning Module

A cleaning module generally comprises any mechanism or system forcleaning a sample holder such as a microplate. A cleaning module (orcleaning function) may be integrated with a fluidics module, forexample, by alternately aspirating and dispensing cleaning and rinsingfluids with the dispense elements. Alternatively, a cleaning module maybe a stand-alone system, for example, having a washer, a dryer, and anoutlet.

The washer is used to wash a sample holder using any suitable mechanismor method. Washing comprises removing sample or other impurities.Typically, the washer cleans a sample holder by spraying, immersing,scrubbing, or otherwise sequentially applying cleaning and rinsingfluids to the sample holder. The sample holder is cleaned of sampleusing the cleaning fluid and rinsed of cleaning fluid (and any residualsample) using the rinsing fluid. The cleaning and rinsing fluids may beidentical. The washer may include apparatus for fluid cleaning such asreservoirs and nozzles, and apparatus for contact cleaning such asscrubbers.

The dryer is used to dry a sample holder using any suitable mechanism ormethod. Drying comprises removing any rinsing fluid remaining afterwashing. Typically, the dryer dries a sample holder using forced air (orother gas), heat, and/or agitation, among others. The dryer may includeapparatus for forcing air such as a gas tank, compressor, and/or nozzle,as well as apparatus for heating and/or agitating such as a heatingelement or spinner. In some systems, drying may consist simply ofroom-temperature air drying.

The outlet is used to eliminate discarded sample, and cleaning andrinsing fluids, using any suitable mechanism or method. Typically, theoutlet comprises a drain and/or a reservoir.

2. Sample-Containment Module

FIGS. 40-43 show a sample-containment module constructed in accordancewith aspects of the invention. The containment module generallycomprises any mechanism or system for sealing wells or other reservoirsin a sample holder. The mechanisms may include temporarily applying asealing sheet to the top of the sample holder and/or removing a sealingsheet prior to dispensing fluid and/or analyzing a sample. The sampleholders including microplates may be of a standard design or of a customdesign especially intended to work with a particular cover. Aspects ofthe invention may include (1) cover materials for sample holders, (2)suitably covered sample holders, (3) systems for automatically applyingand/or removing covers from sample holders, and/or (4) systemsintegrating a sample-containment module with a transport module and/orother function modules.

A sample-containment module may be useful in solving problems associatedwith microplates and other sample holders, particularly forhigh-throughput applications. For example, microplates include wellshaving an open face that permits cross-contamination and evaporation.Therefore, it sometimes is desirable to seal the wells in a microplateto isolate the contents of each well from other wells and from theambient environment. One or both of these functions may be performed inprincipal by a microplate cover. However, microplate covers that havebeen used in the past have problems that make them unsuitable for use ina highly automated laboratory setting. In particular, past microplatecovers may interfere with or prohibit the stacking and/or unstacking ofmicroplates, due to spatial constraints or limitations in the specialhandling equipment used to stack and unstack the microplates. Moreover,microplate covers may permit gas exchange, facilitating evaporation. Inaddition, microplate covers or seals that use adhesive may be difficultto remove or may leave adhesive residue on a surface of a microplate,such that the microplate could stick to other microplates if stacked,unstacked, or otherwise contacted.

FIG. 40 shows a microplate 4110 and a sealing sheet 4112 covering samplewells (not shown) in the microplate. Rectangular sealing sheets 4113 maybe applied to respective microplate tops from an input roll 4114 and/orremoved and transferred from microplate tops to an output roll 4116,where they can be stored prior to disposal. Generally, sealing sheetsmay be stamped and carried on continuous input/output rolls much likeindustry standard labels. The rolls can be loaded into automatedapplication (sealing) and removal (de-sealing) machines configured tooperate with standard and/or specially designed microplates. Stacks ofmicroplates may be fed into application/removal machines, and seals maybe applied or removed as desired. Typically, a sealing sheet will beapplied to a microplate (or other sample holder) before incubationand/or storage, and removed from a microplate before fluidics operationsand/or sample analysis.

Sealing sheets may be designed to include or permit the inclusion ofinformation on the sealing sheet. For example the sealing sheet mayinclude a writeable area and/or a computer readable message or symbolsuch as a barcode.

Sealing sheets also may be designed to control the amount of light thatmay pass through the sheet. A sheet may be substantially opticallytransparent to permit light to pass, for example, so that an opticalanalysis can be performed through the sheet. Alternatively, a sheet maybe substantially optically opaque to prevent light from passing, forexample, to reduce photobleaching. A suitable transparent sealing sheetmay be made of a clear plastic, and a suitable opaque sealing sheet maybe made of aluminum or an aluminized material.

FIG. 41 also shows a microplate 4120 and a sealing sheet 4122 coveringsample wells 4124 in the microplate. Recesses 4128 are formed in aperimeter portion of microplate 4120 to expose edge regions of sealingsheet 4122 to facilitate removal of the sealing sheet by an automated orrobotic device 4130. Robotic device 4130 may include a grippingmechanism and/or picking member for gripping and/or engaging exposededges of sealing sheet 4122. The robotic device may use a number ofdifferent mechanisms to lift sheet 4122 off microplate 4120. Forexample, the robotic device may grab or clamp the edge of sheet 4122 andthen lift the sheet off the microplate. Toward this end, recesses 4128generally comprise any open area (such as notches) adjacent sealingsheet 4122 sufficient to permit grasping and subsequent pulling of thesheet. The robotic device also may pierce and then lift sheet 4122, orapply a vacuum to and then lift sheet 4122. The robotic device also mayapply an adhesive to at least a portion of sheet 4122 before lifting thesheet off microplate 4120.

FIG. 42 is a partial view of an automated sealing-sheet applicationsystem. A conveyor 4150 carries a microplate 4152 in the direction ofarrow 4154 toward a sealing-sheet application site 4156. At applicationsite 4156, a sealing sheet 4158, shown in dashed lines on the undersideof a continuous carrier 4159, is applied precisely over the samplecontainment region of microplate 4152. Successive discrete sealingsheets are carried around a first roller 4160 in the direction of arrow4162. Continuous carrier 4159 is rolled onto a second roller 4163 afterremoval of sealing sheets at site 4156. The materials of microplate4152, sealing sheet 4158, and/or carrier 4159 may be selected so thatsheet 4158 can be reliably pressed onto microplate 4152 and releasedfrom carrier 4159. After applying sealing sheet 4158, conveyor 4150transports sealed microplate 4166 downstream.

FIG. 43 is a partial view of an automated sealing-sheet removal system.A conveyor 4170 transports a sealed microplate 4166 in the direction ofarrow 4172 toward a sealing-sheet removal site 4174. At removal site4174, sheet 4158 is removed from microplate 4152 and transferred to acontinuous carrier 4176, which moves around roller 4178 in the directionof arrow 4180. Uncovered microplate 4152 then moves downstream onconveyor 4170. Transfer of sheets from microplate 4152 to carrier 4176at removal site 4174 may be achieved by selecting a carrier material,optionally containing an adhesive, that can bond to sheet 4158sufficiently to lift the sheet off microplate 4152.

Various mechanical mechanisms may be used to facilitate transfer of asealing sheet onto and off a microplate. For example, a pressure member(e.g., mechanical press) may be applied from above the site. It also maybe helpful to provide Z-height adjustability of carriers 4159 and 4176above conveyors 4150 and 4170. It also may be helpful to provide amechanism for holding the microplate and/or the conveyor when thesealing sheet is being applied and/or removed.

Various mechanical mechanisms also may be used as a conveyor to movemicroplates into and out of sealing-sheet application and removalstations. Such mechanisms include those described above (especially forthe intersite driver) under “Transport Module.”

The sealing-sheet application system and sealing-sheet removal systemmay be configured to enhance flexibility. For example, the systems maybe used alone as stand-alone units or together with one another and/orwith other function modules as part of an integrated system. The systemsalso may be used with a variety of sample holders, including but notlimited to microplates and other sample holders described above under“Sample Holders.”

The invention may provide a microplate sealing system that does notinterfere with the stacking of covered microplates and/or leave anadhesive residue on a perimeter portion of the microplate after thesealing sheet is removed. Adhesive residue remaining on a top side of amicroplate after a sealing sheet is removed may cause stacked plates tostick together, causing a microplate-handling malfunction downstream.These problems may be substantially avoided by carefully controlling thedimension and placement of a sealing sheet on the top surface of amicroplate. Here, the sealing-sheet dimension may be matched to amicroplate so that the sheet substantially covers all of the wells inthe sample containment region of the microplate, while leaving a topperimeter portion of the microplate exposed for contacting anothermicroplate in a stack without interference. For use with standardmicroplates, the sealing sheets may be substantially rectangular, with awidth in the range of 2.75-inches to 3.25-inches, and a length in therange 4.25-inches to 4.75-inches.

3. Sample Incubation Module

FIG. 44 shows an incubation module 4300 constructed in accordance withaspects of the invention. The incubation module generally comprises anymechanism or system for storing or incubating samples with control ofambient environmental conditions, such as temperature, atmosphere (e.g.,humidity, CO₂ level, etc.), agitation, and so on. The mechanisms mayinclude storing the samples in an environmentally controlled enclosureand/or using spacers or other mechanisms to increase thermal and gasexchange around and between samples. An incubation module may be used toprotect thermally sensitive samples such as cells.

Environmental control is especially important with sample holders suchas microplates that may be stacked atop one another before, during,and/or after analysis for transport, incubation, and/or storage.Microplates may be stacked to enhance convenience and minimizefootprint. For example, incubation module 4300 may support a stack ofmicroplates 4302 in the same shelf area used to support a singlemicroplate. Unfortunately, stacking microplates may limit thermal andgas exchange through the space between the microplates and therebycreate gradients in temperature and/or gas composition around thesamples. For example, samples contained in a microplate near the top ofa stack may be exposed to a substantially different temperature or gascomposition than samples contained in microplates near the bottom of thestack.

To facilitate environmental control, the incubation module 4300 mayinclude an enclosure 4304 for storing samples that may be partially ortotally sealed from the outside environment. Moreover, the incubationmodule may include mechanisms for actively controlling the interiorenvironment within the enclosure. For example, incubation module 4300includes a valve device 4306 on a side of the enclosure for controllinggas flow into and out of the enclosure. Suitable gases such as CO₂ maybe injected into the enclosure from a gas source 4308 connected to thevalve and passively or actively removed from the enclosure to facilitatecirculation using a vent 4310, fan 4312, and/or other mechanism.Incubation module 4300 also includes heating and cooling elements 4314in or around the enclosure for raising or lowering the temperatureinside the enclosure. Heating and cooling may be effected from asuitable thermal source 4316. Incubation module 4300 also may includeagitation elements such as a rocker for rocking, shaking, and/orotherwise agitating enclosed sample holders to mix or aerate associatedsamples.

The incubation module may be used in conjunction with other devices andmethods for increasing ambient heat and gas exchange around and betweensamples, especially samples in stacks of sample holders. For example,apertures might be provided in the side of a microplate to allow gas tocirculate in the space between adjacently stacked microplates.Alternatively, or in addition, a spacing mechanism might be providedatop or between microplates to separate a microplate from adjacentlystacked microplates.

FIG. 45 shows a microplate 4350 with adaptations for facilitatingenvironmental access between stacked microplates, with or without anintervening spacer. Microplate 4350 includes a frame portion 4352 and atop portion 4354 having a plurality of sample wells 4356. The frameportion includes apertures 4358 to allow thermal and gas circulationbetween plates. The top portion includes projections 4360 that extendabove a plane defined by the tops of the sample wells to support andelevate a microplate stacked above it, also to allow thermal and gascirculation between plates.

FIG. 46 shows portions of two alternative embodiments of a lid-spacer4400 for facilitating environmental access between stacked microplates.The lid-spacer may be stacked atop, between, or beneath microplates. Thelid-spacer may include a lid portion 4402 and a spacer portion 4404.

Lid portion 4402 provides shape and structural support for thelid-spacer and regulates thermal and/or gas exchange over wells in amicroplate over which the lid-spacer is stacked. The lid portion mayinclude an exterior frame 4406 and a substantially planar cover portion4408 surrounded by the frame portion. The lid portion may fit sealinglyatop a microplate to seal a space over wells in the microplate, reducingevaporation. Alternatively, the lid portion may fit loosely atop amicroplate and/or include apertures 4410 in the frame or operates 4412in the cover portion to permit thermal and/or gas exchange over wells inthe microplate. Alternatively, or in addition, structures such as ridgesmay be provided on the bottom surface of the lid portion to allow gasdiffusion.

Spacer portion 4404 supports and elevates a microplate under which thelid-spacer is stacked, facilitating thermal and/or gas exchange underwells in the microplate. The spacer portion may include projections 4414that extend upward from the lid portion to support a microplate in adesired spaced orientation. These projections typically will be locatedat corners of the lid-spacer to enhance stability, but also may belocated along sides (especially long sides) of the lid-spacer.

The lid-spacer may be formed of any suitable material and manufacturedusing any suitable method. A preferred material is plastic, such as thatused to form microplates. Preferred manufacturing methods includemolding and/or standard machining operations.

The lid-spacer may be sized and shaped to mimic a typical microplate, sothat the lid-spacer can be handled (e.g., singulated) by equipmentdesigned to handle a standard microplate. In this way, the lid-spacermay be singulated or re-stacked by a transport mechanism, and thetransport mechanism can perform de-lidding and re-lidding operations, ifprogrammed to do so. A possible sequence includes the following steps:(1) singulate microplate from input stack, send to function module; (2)singulate lid-spacer from input stack, send to output stack; (3) sendmicroplate from function module to output stack. This sequence can berepeated as desired.

A lid-spacer may be removed and replaced multiple times during assaypreparation, incubation, and/or detection. The lid-spacer may include abar code 4416 or reference fiducial and/or be sized or formed of amaterial permitting sample-handling equipment to distinguish it from amicroplate or other sample holder. The lid-spacer may be transparent topermit light to pass for photoactivations or to perform a luminescenceapplication through the lid-spacer. Alternatively, the lid-spacer may beopaque to preserve darkness inside the wells to prevent photobleachingprior to a luminescence application. Lid-spacers can be placed at thetop and bottom of a stack to ensure that plates are always covered asplates are circulated back and forth in stacks during assay preparation,incubation, and/or detection. A stack of microplates with respectivelid-spacers may be transported in a magazine back and forth between anincubator, detector, fluid dispenser, and/or transporter when long-termincubations in a controlled environment are required.

FIG. 47 shows a stack 4450 of microplates 4452 and intervening spacingdevices 4454. Spacing device 4454 is substantially the same as thelid-spacer shown in FIG. 46. A singulation latch 4456 operates on thebottom of the stack to remove one microplate or one spacing device at atime.

The incubation module may be used alone as a stand-alone unit ortogether with one or more other function modules as part of anintegrated system. FIG. 48 shows such an integrated system 4500, whichmay be used to process and analyze a sample contained in a microplate.System 4500 includes a microplate input site 4502, a fluidics module4504, an incubation module 4506, and an analysis module 4508. Atransport module 4510 transports microplates from site to site. Amicroplate 4512 is singulated from the bottom of a stack in input site4502. Transport module 4510 transports the microplate to fluidics module4504, where fluid is added to wells in the microplate. The microplate4512 is then transported and stacked in incubation module 4506, alongwith other microplates and intervening spacers and lids in accordancewith previously described embodiments of the invention. After thesamples are incubated in incubation module 4506, the microplate may besingulated from the bottom of a stack in the incubation module andtransported to analysis module 4508 where a test is performed on thesample.

The lid-spacer may be combined advantageously with a microplate sealer.It also may be advantageous to employ semipermeable films or membranesselectively to control environmental access to samples contained inwells under a spacer.

E. Analysis Module

The transport module, fluidics module, and/or auxiliary modulesdescribed in the preceding sections may be combined with an analysismodule to form an integrated system for sample preparation and/oranalysis. An analysis module generally comprises any mechanism or systemfor analyzing a sample, including qualitative analysis (to determine thenature of the sample and/or its components) and/or quantitative analysis(to determine the amount, relative proportions, and/or activity of thesample and/or its components).

FIG. 49 shows a system 5050 for analyzing samples that includes atransport module 5052 and an exemplary analysis module 5054 capable ofdetecting and analyzing light. The transport module includes I/O sites5056, a transfer site 5058, and mechanisms (not visible) fortransporting sample holders between the I/O and transfer sites, asdescribed above. The analysis module includes a housing 5060, a moveablecontrol unit 5062, an optical system (not visible), and a transportmechanism 5064. The housing may be used to enclose the analysis module,protecting both the user and components of the module. The control unitmay be used to operate the module manually and/or robotically, asdescribed in U.S. Pat. No. 6,025,985, which is incorporated herein byreference. The optical system and transport mechanisms are described insubsequent sections.

The analysis and transport modules may be configured so that transportmechanism 5064 from analysis module 5054 can interact at transfer site5058 with an intrasite driver (not visible) from transport module 5052for sample transfer. More specifically, the transport mechanism mayinteract with an intrasite driver from the transport module, such asintrasite driver 2300 and lifters 2308 b in FIG. 23.

Further aspects of the analysis module are presented in the followingsections: (1) optical system, (2) transport mechanism, and (3)analytical methods.

1. Optical System

FIGS. 50-53 show an optical system (and related components) 5090 for usein system 5050. The optical system may include components for generatingand/or detecting light, and for transmitting light to and/or from asample. These components may include (1) a stage for supporting thesample, (2) one or more light sources for delivering light to thesample, (3) one or more detectors for receiving light transmitted fromthe sample and converting it to a signal, (4) first and second opticalrelay structures for relaying light between the light source, sample,and detector, and/or (5) a processor for analyzing the signal from thedetector. Module components may be chosen to optimize speed,sensitivity, and/or dynamic range for one or more assays. For example,optical components with low intrinsic luminescence may be used toenhance sensitivity in luminescence assays by reducing background.Module components also may be shared by different assays, or dedicatedto particular assays. For example, steady-state photoluminescence assaysmay use a continuous light source, time-resolved photoluminescenceassays may use a time-varying light source, and chemiluminescence assaysmay not use a light source. Similarly, steady-state and time-resolvedphotoluminescence assays may both use a first detector, andchemiluminescence assays may use a second detector.

Optical system 5090 includes (a) a photoluminescence optical system, and(b) a chemiluminescence optical system, as described below. Furtheraspects of the optical system are described in the following patentapplications, which are incorporated herein by reference: U.S. patentapplication Ser. No. 09/160,533, filed Sep. 24, 1998; U.S. patentapplication Ser. No. 09/349,733, filed Jul. 8, 1999; PCT PatentApplication Ser. No. PCT/US99/16287, filed Jul. 26, 1999; and PCT PatentApplication Ser. No. PCT/US00/04543, filed Feb. 22, 2000.

a. Photoluminescence Optical System

FIGS. 50-52 show the photoluminescence (or incident light-based) opticalsystem of optical system 5090. As configured here, optical system 5090includes a continuous light source 5100 and a time-modulated lightsource 5102. Optical system 5090 includes light source slots 5103 a-dfor four light sources, although other numbers of light source slots andlight sources also could be provided. Light source slots 5103 a-dfunction as housings that may surround at least a portion of each lightsource, providing some protection from radiation and explosion. Thedirection of light transmission through the incident light-based opticalsystem is indicated by arrows.

Continuous source 5100 provides light for absorbance, scattering,photoluminescence intensity, and steady-state photoluminescencepolarization assays. Continuous light source 5100 may include arc lamps,incandescent lamps, fluorescent lamps, electroluminescent devices,lasers, laser diodes, and light-emitting diodes (LEDs), among others. Apreferred continuous source is a high-intensity, high color temperaturexenon arc lamp, such as a Model LX175F CERMAX xenon lamp from ILCTechnology, Inc. Color temperature is the absolute temperature in Kelvinat which a blackbody radiator must be operated to have a chromaticityequal to that of the light source. A high color temperature lampproduces more light than a low color temperature lamp, and it may have amaximum output shifted toward or into visible wavelengths andultraviolet wavelengths where many luminophores absorb. The preferredcontinuous source has a color temperature of 5600 Kelvin, greatlyexceeding the color temperature of about 3000 Kelvin for a tungstenfilament source. The preferred source provides more light per unit timethan flash sources, averaged over the flash source duty cycle,increasing sensitivity and reducing read times. Optical system 5090 mayinclude a modulator mechanism configured to vary the intensity of lightincident on the sample without varying the intensity of light producedby the light source. Further aspects of the continuous light source aredescribed in U.S. patent application Ser. No. 09/349,733, filed Jul. 8,1999, which is incorporated herein by reference.

Time-modulated source 5102 provides light for time-resolved absorbanceand/or photoluminescence assays, such as photoluminescence lifetime andtime-resolved photoluminescence polarization assays. A preferredtime-modulated source is a xenon flash lamp, such as a Model FX-1160xenon flash lamp from EG&G Electro-Optics. The preferred source producesa “flash” of light for a brief interval before signal detection and isespecially well suited for time-domain measurements. Othertime-modulated sources include pulsed lasers, electronically modulatedlasers and LEDs, and continuous lamps and other sources whose intensitycan be modulated extrinsically using a Pockels cell, Kerr cell, or othermechanism. Such other mechanisms may include an amplitude modulator suchas a chopper as described in PCT Patent Application Ser. No.PCT/US99/16287, filed Jul. 26, 1999, which is incorporated herein byreference. Extrinsically modulated continuous light sources areespecially well suited for frequency-domain measurements.

In optical system 5090, continuous source 5100 and time-modulated source5102 produce multichromatic, unpolarized, and incoherent light.Continuous source 5100 produces substantially continuous illumination,whereas time-modulated source 5102 produces time-modulated illumination.Light from these light sources may be delivered to the sample withoutmodification, or it may be filtered to alter its intensity, spectrum,polarization, or other properties.

Light produced by the light sources follows an excitation optical pathto an examination site or measurement region. Such light may passthrough one or more “spectral filters,” which generally comprise anymechanism for altering the spectrum of light that is delivered to thesample. Spectrum refers to the wavelength composition of light. Aspectral filter may be used to convert white or multichromatic light,which includes light of many colors, into red, blue, green, or othersubstantially monochromatic light, which includes light of one or only afew colors. In optical system 5090, spectrum is altered by an excitationinterference filter 5104, which preferentially transmits light ofpreselected wavelengths and preferentially absorbs light of otherwavelengths. For convenience, excitation interference filters 5104 maybe housed in an excitation filter wheel 5106, which allows the spectrumof excitation light to be changed by rotating a preselected filter intothe optical path. Spectral filters also may separate light spatially bywavelength. Examples include gratings, monochromators, and prisms.

Spectral filters are not required for monochromatic (“single color”)light sources, such as certain lasers, which output light of only asingle wavelength. Therefore, excitation filter wheel 5106 may bemounted in the optical path of some light source slots 5103 a,b, but notother light source slots 5103 c,d. Alternatively, the filter wheel mayinclude a blank station that does not affect light passage.

Light next passes through an excitation optical shuttle (or switch)5108, which positions an excitation fiber optic cable 5110 a,b in frontof the appropriate light source to deliver light to top or bottom opticsheads 5112 a,b, respectively. Light is transmitted through a fiber opticcable much like water is transmitted through a garden hose. Fiber opticcables can be used easily to turn light around comers and to route lightaround opaque components of the apparatus. Moreover, fiber optic cablesgive the light a more uniform intensity profile. A preferred fiber opticcable is a fused silicon bundle, which has low autoluminescence. Despitethese advantages, light also can be delivered to the optics heads usingother mechanisms, such as mirrors.

Light arriving at the optics head may pass through one or moreexcitation “polarization filters,” which generally comprise anymechanism for altering the polarization of light. Excitationpolarization filters may be included with the top and/or bottom opticshead. In optical system 5090, polarization is altered by excitationpolarizers 5114, which are included only with top optics head 5112 a fortop reading; however, such polarizers also can be included with bottomoptics head 5112 b for bottom reading. Excitation polarization filters5114 may include an s-polarizer S that passes only s-polarized light, ap-polarizer P that passes only p-polarized light, and a blank O thatpasses substantially all light, where polarizations are measuredrelative to the beamsplitter. Excitation polarizers 5114 also mayinclude a standard or ferro-electric liquid crystal display (LCD)polarization switching system. Such a system may be faster than amechanical switcher. Excitation polarizers 5114 also may include acontinuous mode LCD polarization rotator with synchronous detection toincrease the signal-to-noise ratio in polarization assays. Excitationpolarizers 5114 may be incorporated as an inherent component in somelight sources, such as certain lasers, that intrinsically producepolarized light. Further aspects of the polarization filters and theiruse in polarization assay are described in U.S. patent application Ser.No. 09/349,733, filed Jul. 8, 1999, which is incorporated herein byreference.

Light at one or both optics heads also may pass through an excitation“confocal optics element,” which generally comprises any mechanism forfocusing light into a “sensed volume.” In optical system 5090, theconfocal optics element includes a set of lenses 5117 a-c and anexcitation aperture 5116 placed in an image plane conjugate to thesensed volume, as shown in FIG. 52. Aperture 5116 may be implementeddirectly, as an aperture, or indirectly, as the end of a fiber opticcable. Preferred apertures have diameters of 1 mm and 1.5 mm. Lenses5117 a,b project an image of aperture 5116 onto the sample, so that onlya preselected or sensed volume of the sample is illuminated. The area ofillumination will have a diameter corresponding to the diameter of theexcitation aperture.

Light traveling through the optics head is directed onto a beamsplitter5118, which reflects light toward a sample 5120 and transmits lighttoward a light monitor 5122. The reflected light passes through lens5117 b, which is operatively positioned between beamsplitter 5118 andsample 5120.

Beamsplitter 5118 is used to direct excitation or incident light towardthe sample and light monitor, and to direct light leaving the sampletoward the detector. The beamsplitter is changeable, so that it may beoptimized for different assay modes or samples. In some embodiments,switching between beamsplitters may be performed manually, whereas inother embodiments, such switching may be performed automatically.Automatic switching may be performed based on direct operator command,or based on an analysis of the sample by the instrument. If a largenumber or variety of photoactive molecules are to be studied, thebeamsplitter must be able to accommodate light of many wavelengths; inthis case, a “50:50” beamsplitter that reflects half and transmits halfof the incident light independent of wavelength is optimal. Such abeamsplitter can be used with many types of molecules, while stilldelivering considerable excitation light onto the sample, and whilestill transmitting considerable light leaving the sample to thedetector. If one or a few related photoactive molecules are to bestudied, the beamsplitter needs only to be able to accommodate light ata limited number of wavelengths; in this case, a “dichroic” or“multidichroic” beamsplitter is optimal. Such a beamsplitter can bedesigned with cutoff wavelengths for the appropriate sets of moleculesand will reflect most or substantially all of the excitation andbackground light, while transmitting most or substantially all of theemission light in the case of luminescence. This is possible because thebeamsplitter may have a reflectivity and transmissivity that varies withwavelength.

The beamsplitter more generally comprises any optical device fordividing a beam of light into two or more separate beams. A simplebeamsplitter (such as a 50:50 beamsplitter) may include a very thinsheet of glass inserted in the beam at an angle, so that a portion ofthe beam is transmitted in a first direction and a portion of the beamis reflected in a different second direction. A more sophisticatedbeamsplitter (such as a dichroic or multi-dichroic beamsplitter) mayinclude other prismatic materials, such as fused silica or quartz, andmay be coated with a metallic or dielectric layer having the desiredtransmission and reflection properties, including dichroic andmulti-dichroic transmission and reflection properties. In somebeamsplitters, two right-angle prisms are cemented together at theirhypotenuse faces, and a suitable coating is included on one of thecemented faces. Further aspects of the beamsplitter are described in PCTPatent Application Serial No. PCT/US00/06841, filed Mar. 15, 2000, whichis incorporated herein by reference.

Light monitor 5122 is used to correct for fluctuations in the intensityof light provided by the light sources. Such corrections may beperformed by reporting detected intensities as a ratio overcorresponding times of the luminescence intensity measured by thedetector to the excitation light intensity measured by the lightmonitor. The light monitor also can be programmed to alert the user ifthe light source fails. A preferred light monitor is a siliconphotodiode with a quartz window for low autoluminescence.

The sample (or composition) may be held in a sample holder supported bya stage 5123. The sample can include compounds, mixtures, surfaces,solutions, emulsions, suspensions, cell cultures, fermentation cultures,cells, tissues, secretions, and/or derivatives and/or extracts thereof.Analysis of the sample may involve measuring the presence,concentration, or physical properties (including interactions) of aphotoactive analyte in such a sample. Sample may refer to the contentsof a single microplate well, or several microplate wells, depending onthe assay. In some embodiments, such as a portable apparatus, the stagemay be intrinsic to the instrument.

The sample holder can include microplates, biochips, or any arrangementof samples in a known format, as described above. In optical system5090, the preferred sample holder is a microplate 5124, which includes aplurality of microplate wells 5126 for holding samples. Microplates aretypically substantially rectangular holders that include a plurality ofsample wells for holding a corresponding plurality of samples. Thesesample wells are normally cylindrical in shape although rectangular orother shaped wells are sometimes used. The sample wells are typicallydisposed in regular arrays. The “standard” microplate includes 96cylindrical sample wells disposed in a 8×12 rectangular array on 9millimeter centers.

The sensed volume typically has an hourglass shape, with a cone angle ofabout 25° and a minimum diameter ranging between 0.1 mm and 2.0 mm. For96-well and 384-well microplates, a preferred minimum diameter is about1.5 mm. For 1536-well microplates, a preferred minimum diameter is about1.0 mm. The size and shape of the sample holder may be matched to thesize and shape of the sensed volume, as described in U.S. patentapplication Ser. No. 09/062,472, filed Apr. 17, 1998, which isincorporated herein by reference.

The position of the sensed volume can be moved precisely within thesample to optimize the signal-to-noise and signal-to-background ratios.For example, the sensed volume may be moved away from walls in thesample holder to optimize signal-to-noise and signal-to-backgroundratios, reducing spurious signals that might arise from luminophoresbound to the walls and thereby immobilized. In optical system 5090,position in the X,Y-plane perpendicular to the optical path iscontrolled by moving the stage supporting the sample, whereas positionalong the Z-axis parallel to the optical path is controlled by movingthe optics heads using a Z-axis adjustment mechanism 5130, as shown inFIGS. 50 and 51. However, any mechanism for bringing the sensed volumeinto register or alignment with the appropriate portion of the samplealso may be employed. For example, the optics head also may be scannedin the X,Y-plane, as described in U.S. Provisional Patent ApplicationSer. No. 60/142,721, filed Jul. 7, 1999, which is incorporated herein byreference.

The combination of top and bottom optics permits assays to combine: (1)top illumination and top detection, or (2) top illumination and bottomdetection, or (3) bottom illumination and top detection, or (4) bottomillumination and bottom detection. Same-side illumination and detection,(1) and (4), is referred to as “epi” and is preferred forphotoluminescence and scattering assays. Opposite-side illumination anddetection, (2) and (3), is referred to as “trans” and has been used inthe past for absorbance assays. In optical system 5090, epi modes aresupported, so the excitation and emission light travel the same path inthe optics head, albeit in opposite or anti-parallel directions.However, trans modes also can be used with additional sensors, asdescribed below. In optical system 5090, top and bottom optics headsmove together and share a common focal plane. However, in otherembodiments, top and bottom optics heads may move independently, so thateach can focus independently on the same or different sample planes.

Generally, top optics can be used with any sample holder having an opentop, whereas bottom optics can be used only with sample holders havingoptically transparent bottoms, such as glass or thin plastic bottoms.Clear bottom sample holders are particularly suited for measurementsinvolving analytes that accumulate on the bottom of the holder.

Light may be transmitted by the sample in multiple directions. A portionof the transmitted light will follow an emission pathway to a detector.Transmitted light passes through lens 5117 c and may pass through anemission aperture 5131 and/or an emission polarizer 5132. In opticalsystem 5090, the emission aperture is placed in an image plane conjugateto the sensed volume and transmits light substantially exclusively fromthis sensed volume. In optical system 5090, the emission apertures inthe top and bottom optical systems are the same size as the associatedexcitation apertures, although other sizes also may be used. Theemission polarizers are included only with top optics head 5112 a. Theemission aperture and emission polarizer are substantially similar totheir excitation counterparts. Emission polarizer 5132 may be includedin detectors that intrinsically detect the polarization of light.

Excitation polarizers 5114 and emission polarizers 5132 may be usedtogether in nonpolarization assays to reject certain background signals.Luminescence from the sample holder and from luminescent moleculesadhered to the sample holder is expected to be polarized, because therotational mobility of these molecules should be hindered. Suchpolarized background signals can be eliminated by “crossing” theexcitation and emission polarizers, that is, setting the angle betweentheir transmission axes at 90°. As described above, such polarizedbackground signals also can be reduced by moving the sensed volume awayfrom walls of the sample holder. To increase signal level, beamsplitter5118 should be optimized for reflection of one polarization andtransmission of the other polarization. This method will work best wherethe luminescent molecules of interest emit relatively unpolarized light,as will be true for small luminescent molecules in solution.

Transmitted light next passes through an emission fiber optic cable 5134a,b to an emission optical shuttle (or switch) 5136. This shuttlepositions the appropriate emission fiber optic cable in front of theappropriate detector. In optical system 5090, these components aresubstantially similar to their excitation counterparts, although othermechanisms also could be employed.

Light exiting the fiber optic cable next may pass through one or moreemission “intensity filters,” which generally comprise any mechanism forreducing the intensity of light. Intensity refers to the amount of lightper unit area per unit time. In optical system 5090, intensity isaltered by emission neutral density filters 5138, which absorb lightsubstantially independent of its wavelength, dissipating the absorbedenergy as heat. Emission neutral density filters 5138 may include ahigh-density filter H that absorbs most incident light, a medium-densityfilter M that absorbs somewhat less incident light, and a blank O thatabsorbs substantially no incident light. These filters may be changedmanually, or they may be changed automatically, for example, by using afilter wheel. Intensity filters also may divert a portion of the lightaway from the sample without absorption. Examples include beamsplitters, which transmit some light along one path and reflect otherlight along another path, and diffractive beam splitters (e.g.,acousto-optic modulators), which deflect light along different pathsthrough diffraction. Examples also include hot mirrors or windows thattransmit light of some wavelengths and absorb light of otherwavelengths.

Light next may pass through an emission interference filter 5140, whichmay be housed in an emission filter wheel 5142. In optical system 5090,these components are substantially similar to their excitationcounterparts, although other mechanisms also could be employed. Emissioninterference filters block stray excitation light, which may enter theemission path through various mechanisms, including reflection andscattering. If unblocked, such stray excitation light could be detectedand misidentified as photoluminescence, decreasing thesignal-to-background ratio. Emission interference filters can separatephotoluminescence from excitation light because photoluminescence haslonger wavelengths than the associated excitation light. Luminescencetypically has wavelengths between 200 and 2000 nanometers.

The relative positions of the spectral, intensity, polarization, andother filters presented in this description may be varied withoutdeparting from the spirit of the invention. For example, filters usedhere in only one optical path, such as intensity filters, also may beused in other optical paths. In addition, filters used here in only topor bottom optics, such as polarization filters, may also be used in theother of top or bottom optics or in both top and bottom optics. Theoptimal positions and combinations of filters for a particularexperiment will depend on the assay mode and the sample, among otherfactors.

Light last passes to a detector, which is used in absorbance, scatteringand photoluminescence assays, among others. In optical system 5090,there is one detector 5144, which detects light from all modes. Apreferred detector is a photomultiplier tube (PMT). Optical system 5090includes detector slots 5145 a-d for four detectors, although othernumbers of detector slots and detectors also could be provided.

More generally, detectors comprise any mechanism capable of convertingenergy from detected light into signals that may be processed by theapparatus, and by the processor in particular. Suitable detectorsinclude photomultiplier tubes, photodiodes, avalanche photodiodes,charge-coupled devices (CCDs), and intensified CCDs, among others.Depending on the detector, light source, and assay mode, such detectorsmay be used in a variety of detection modes. These detection modesinclude (1) discrete (e.g., photon-counting) modes, (2) analog (e.g.,current-integration) modes, and/or (3) imaging modes, among others, asdescribed in PCT Patent Application Ser. No. PCT/US99/03678.

b. Chemiluminescence Optical System

FIGS. 50, 51, and 53 show the chemiluminescence optical system ofoptical system 5090. Because chemiluminescence follows a chemical eventrather than the absorption of light, the chemiluminescence opticalsystem does not require a light source or other excitation opticalcomponents. Instead, the chemiluminescence optical system requires onlyselected emission optical components. In optical system 5090, a separatelensless chemiluminescence optical system is employed, which isoptimized for maximum sensitivity in the detection of chemiluminescence.

Generally, components of the chemiluminescence optical system performthe same functions and are subject to the same caveats and alternativesas their counterparts in the incident light-based optical system. Thechemiluminescence optical system also can be used for other assay modesthat do not require illumination, such as electrochemiluminescence.

The chemiluminescence optical path begins with a chemiluminescent sample5120 held in a sample holder 5126. The sample and sample holder areanalogous to those used in photoluminescence assays; however, analysisof the sample involves measuring the intensity of light generated by achemiluminescence reaction within the sample rather than bylight-induced photoluminescence. A familiar example of chemiluminescenceis the glow of the firefly.

Chemiluminescence light typically is transmitted from the sample in alldirections, although most will be absorbed or reflected by the walls ofthe sample holder. A portion of the light transmitted through the top ofthe well is collected using a chemiluminescence head 5150, as shown inFIG. 50, and will follow a chemiluminescence optical pathway to adetector. The direction of light transmission through thechemiluminescence optical system is indicated by arrows.

The chemiluminescence head includes a nonconfocal mechanism fortransmitting light from a sensed volume within the sample. Detectingfrom a sensed volume reduces contributions to the chemiluminescencesignal resulting from “cross talk,” which is pickup from neighboringwells. The nonconfocal mechanism includes a chemiluminescence baffle5152, which includes rugosities 5153 that absorb or reflect light fromother wells. The nonconfocal mechanism also includes a chemiluminescenceaperture 5154 that further confines detection to a sensed volume.

Light next passes through a chemiluminescence fiber optic cable 5156,which may be replaced by any suitable mechanism for directing light fromthe sample toward the detector. Fiber optic cable 5156 is analogous toexcitation and emission fiber optic cables 5110 a,b and 5134 a,b in thephotoluminescence optical system. Fiber optic cable 5156 may include atransparent, open-ended lumen that may be filled with fluid. This lumenwould allow the fiber optic to be used both to transmit luminescencefrom a microplate well and to dispense fluids into the microplate well.The effect of such a lumen on the optical properties of the fiber opticcould be minimized by employing transparent fluids having opticalindices matched to the optical index of the fiber optic.

Light next passes through one or more chemiluminescence intensityfilters, which generally comprise any mechanism for reducing theintensity of light. In optical system 5090, intensity is altered bychemiluminescence neutral density filters 5158. Light also may passthrough other filters, if desired.

Light last passes to a detector, which converts light into signals thatmay be processed by the apparatus. In optical system 5090, there is onechemiluminescence detector 5160. This detector may be selected tooptimize detection of blue/green light, which is the type most oftenproduced in chemiluminescence. A preferred detection is aphotomultiplier tube, selected for high quantum efficiency and low darkcount at chemiluminescence wavelengths (400-500 nanometers).

2. Transport Mechanism

FIGS. 54-57 show a transport mechanism or stage, which generallycomprises any mechanism for supporting a sample in a sample holder foranalysis by an analysis module. (The transport module also may be usedto support a sample for fluid dispensing, as described above.) Inanalysis module 5054, the stage includes a transporter 5600 and baseplatform 5700.

FIGS. 54-56 show transporter 5600, which includes a transporter body5602 and substantially parallel first and second transporter flanges5604 a,b that extend outward from transporter body 5602. First andsecond transporter flanges 5604 a,b terminate in first and secondtransporter extensions 5606 a,b that turn in toward one another withoutcontacting one another. Transporter extensions 5606 a,b may be joined bya connector portion 5607. Transporter body 5602, flanges 5604 a,b, andextensions 5606 a,b lie substantially in a plane and define atransporter cavity 5608 that is larger than the expected peripheraldimension of any sample holders which the transporter is intended tosupport. The shape of this cavity is chosen to accommodate the shape ofthe preferred sample holders. In analysis module 5054, cavity 5608 isgenerally rectangular to accommodate generally rectangular sampleholders, such as microplates. In analysis module 5054, long sides of therectangular sample holder are positioned against flanges 5604 a,b.

Transporter 5600 includes a shelf structure and associated framestructure for supporting a microplate or other sample holder. Forexample, transporter shelves 5610 along portions of body 5602, flanges5604 a,b, and extensions 5606 a,b form a shelf structure that supportsthe bottom of the sample holder. The shelf structure also could includeother support mechanisms, such as pins or pegs.

The transporter also includes an automatic sample holder positioningmechanism 5620 for positioning sample holders precisely and reproduciblywithin cavity 5608. Mechanism 5620 includes Y and X axis positioningarms 5622 a,b that contact the sample holder to control its Y and Xposition, respectively. Here, a Y axis is defined as generally parallelto transporter flanges 5604 a,b, and an X axis is defined asperpendicular to the Y axis and generally parallel to transporterextensions 5606 a,b. Other coordinate systems also can be defined, solong as they include two noncolinear directions.

Y-axis positioning arm 5622 a lies substantially within a channel 5624in body 5602. Y-axis positioning arm 5622 a includes a rod 5626 a, whichis bent at substantially right angles to form three substantiallycoplanar and equal-lengthed segments. A first end segment 5628 a of rod5626 a terminates near cavity 5608 in a bumper 5632 for engaging asample holder. A second end segment 5634 a of rod 5626 a terminates awayfrom cavity 5608 in an actuator tab 5636 a for controlling movement ofarm 5622 a. Actuator tab 5636 a is bent away from body 5602. First andsecond end segments 5628 a, 5634 a are substantially parallel. A middlesegment 5638 a of rod 5626 a connects the two end segments at theirnontabbed ends 5640, 5641. An X-axis biasing spring 5642 a having firstand second spring ends 5644, 5648 is slipped over rod 5626 a. Firstspring end 5644 is held to second end segment 5634 a of rod 5626 a by aclamping-type retaining ring 5650. Second spring end 5648 rests againsta rod bearing 5652. The Y-axis biasing spring extends substantiallyparallel to first and second end segments 5628 a, 5634 a. The force fromspring 5642 a is transmitted to rod 5626 a by the clamping action ofretaining ring 5650.

X-axis positioning arm 5622 b also lies substantially within channel5624 in body 5602 and is similar to Y-axis positioning arm, except that(1) first end segment 5628 b is longer and middle segment 5638 b isshorter in rod 5626 b of the X-axis positioning arm than in rod 5626 aof the Y-axis positioning arm, (2) first end segment 5628 a terminatesin a lever tab 5653 in the X-axis positioning arm rather than in bumper5632 in the Y-axis positioning arm, and (3) the two rods bend inopposite directions between first end segments 5628 a,b and second endsegments 5634 a,b.

X-axis positioning arm 5622 b is connected via lever tab 5653 to anX-axis positioning lever 5654 that lies along transporter flange 5604 b.X-axis positioning lever 5654 includes first and second leverprojections 5656, 5658 and is pivotally mounted about a lever pivot axis5659 to transporter 5600 near the intersection of body 5602 and flange5604 b. First lever projection 5656 is substantially perpendicular toflange 5604 b and abuts lever tab 5630 b on X-axis positioning arm 5622b for actuating the positioning lever. Second lever projection 5658 alsois substantially perpendicular to flange 5604 b and includes an edge5660 for contacting a sample holder.

Transporter 5600 functions as follows. For loading, the transporteroccupies a loading position substantially outside a housing. In thisposition, actuator tabs 5636 a,b abut an actuator bar 5670, shown inFIG. 57. In addition, biasing springs 5642 a,b are compressed, andbumper 5632 and second projection 5658 having edge 5660 are pulled outof cavity 5608. A person, robot, or mechanical stacker then can place asample holder into cavity 5608 so that the bottom of the sample holderrests on shelves 5610. Cavity 5608 is larger than the sample holder tofacilitate this placement and to accommodate variations in sample holdersize.

In some configurations, connector portion 5607 may be removed, such thattransporter 5600 has an open end. This open end permits a microplatetransfer device to enter cavity 5608 and the generally rectangular areaof the holder. The microplate transfer device may, after moving into thegenerally rectangular area, move down relative to transporter 5600,thereby gently placing the microplate into the generally rectangulararea.

For reading, the transporter must deliver the sample holder to anexamination site inside the housing. In this process, the transportermoves parallel to second end segments 5634 a,b, and actuator tabs 5636a,b disengage actuator bar 5670. Biasing spring 5642 a pushes Y-axispositioning arm 5622 a toward cavity 5608. Bumper 5632 engages thesample holder and pushes it away from body 5602 until it abutsextensions 5606 a,b. Biasing spring 5642 b pushes X-axis positioning arm5622 b toward cavity 5608. Edge 5660 of second projection 5658 engagesthe sample holder and pushes it away from flange 5604 b until it abutsflange 5604 a.

As long as the sample holder is placed in any position on the lowerguide shelves, it may be positioned (registered) precisely andreproducibly against a reference corner 5672 within cavity 5608 underthe action of both positioning arms. Biasing springs 5642 a,b can bechosen to have different strengths, so that the X-Y positioning actionis performed less or more forcefully. In analysis module 5054, middlesegment 5638 b and first lever projection 5656 of positioning lever 5654can be varied in length to cause registration to occur in series, firstalong the X-axis or first along the Y-axis, and second along the Y-axisor second along the X-axis, respectively. For example, reducing thelength of middle segment 5638 b and reducing the length of projection5656 will cause registration to occur first in the X-axis, and second inthe Y-axis.

Positioning lever 5654 and bumper 5632 are retracted when body 5602 ofthe automatic microplate positioning transporter is moved to the ejectposition by the X,Y stage. Thus, the microplate is placed on transportershelf 5610 only when the lever and bumper are retracted. Two springs5642 a,b are attached to the rods, which run along the length of thetransporter body and end perpendicular to the body. When the transporteris moved to the eject position, the two perpendicular ends of the rodsencounter a stop 5670, which consists of a rectangular structure locatedabove and parallel to the body. The stop prevents the two perpendicularends of the actuators, and thus the actuators, from moving with thetransporter body. This causes the two springs to contract, changing theposition of the transporter arms and increasing the amount of room forthe microplate. The microplate then can be placed on the guide shelf ofthe body. When the body of the automatic microplate positioningtransporter is moved back away from the stop, the two perpendicular endsof the actuators no longer are blocked, which allows the actuators,springs, and transporter arms to move into their original position. Theexpansion of the springs pushes the microplate exactly into position, asdefined by the reference comer.

Thus, components of transporter 5600 act as first and second releasableclamp mechanisms. The first releasable clamp mechanism applies a forceagainst a first (e.g., Y or X) side of the microplate, thereby securingthe microplate in the holder. The second releasable clamp mechanismapplies a force against a second (e.g., X or Y) side of the microplate,thereby securing the microplate in the holder from two sides. Theseclamp mechanisms may sandwich a microplate between the positioning armsand opposing portions of the frame structure, such that the positioningarms function as pushers and the opposing portions of the framestructure function as bumpers for the clamp mechanisms.

The invention provides a method of automatically feeding microplates inand out of an analyzer. The method comprises (1) automaticallydelivering a microplate just outside an opening to the analyzer, (2)moving a gripping device from inside the analyzer, through the opening,to a location immediately below the microplate; and (3) gently placingthe microplate onto the gripping device. The method further may compriseclamping the microplate in the holder by applying a first force againsta first side of the microplate, applying a second force against a secondside of the microplate, and/or serially performing the clamping steps.

FIG. 57 shows a base platform 5700 with drive mechanisms for moving atransporter 5702 between loading and examination positions or sites. Aspreviously described, transporter 5702 includes flanges 5704 a,bdefining a cavity 5706 for receiving and gripping a microplate (notshown). A Y-axis drive mechanism 5707 is provided for moving transporter5702 along a first track 5708 relative to the Y-axis, from a loadingposition 5710 toward an examination position 5712. An X-axis drivemechanism 5713 is provided to move transporter 5702 to examinationposition 5712 along a second track 5714 relative to the X-axis.

In operation, a microplate is loaded in transporter 5702 at loadingposition 5710. This loading position may correspond to the transfer sitefor a transport module, as shown in FIG. 49. Transporter 5702 is thendriven toward the examination site (and/or optional fluid dispense site5716) by Y-axis drive mechanism 5707. A sensor (not shown) detects thepresence of the sample holder. The analyzer may be configuredautomatically to read the microplate once the sensor detects itspresence, or the analyzer may be configured to signal the systemcontroller through a data port that a microplate has been received andthat the analyzer is ready to accept a command to begin reading. The X-and Y-axis drive mechanisms then operate together to align selectedmicroplate wells with an optical axis, substantially parallel to aZ-axis, along which a sensed volume for luminescence detection may bedefined by optical components contained in one or both of a top andbottom optics head positioned above and below base platform 5700,respectively.

Transporter 5700 thus may function both as a sample delivery device inand out of the analyzer, and as a moveable stage for supporting thesample holder at the examination site (and/or at the optional fluiddispense site). The cavity in the transporter permits analysis to becarried out from below the holder, when the transporter is functioningas a stage at the examination site.

X- and Y-axis drive mechanisms 5707 and 5713 may be controlled by ahigh-performance motion control system that maximizes throughput whileminimizing detection errors. A preferred high-performance control systemincludes precision five-phase stepper motors that employ encoderfeedback to move the microplate quickly and accurately to each readposition. The control system may optimize the acceleration/decelerationprofiles of the microplate to minimize shaking of fluid within themicroplate, for example, by minimizing “jerk” (the time rate of changeof the acceleration of the microplate). Alternatively, the controlsystem may increase throughput by moving plates more quickly, if highervariation in results due to increased shaking and settling time may betolerated.

3. Analytical Methods

Analysis modules may be used to analyze a sample, qualitatively orquantitatively, as described above. Suitable methods for such analysismay include spectroscopic, hydrodynamic, and imaging methods, amongothers, especially those adaptable to high-throughput analysis ofmultiple samples.

Spectroscopic methods may involve interaction of light (or wavelikeparticles) with matter, and may involve monitoring some property of thelight that is changed due to the interaction. Suitable spectroscopicmethods may include absorption, luminescence (includingphotoluminescence, chemiluminescence, and electrochemiluminescence),magnetic resonance (including nuclear and electron spin resonance),scattering (including light scattering, electron scattering, and neutronscattering), circular dichroism, and optical rotation, among others.Suitable photoluminescence methods may include fluorescence intensity(FLINT), fluorescence polarization (FP), fluorescence resonance energytransfer (FRET), fluorescence lifetime (FLT), total internal reflectionfluorescence (TIRF), fluorescence correlation spectroscopy (FCS), andfluorescence recovery after photobleaching (FRAP), as well as theirphosphorescence and higher-order-transition analogs, among others.

Hydrodynamic methods may involve interaction of a molecule or othercompound with its neighbors, its solvent, and/or a matrix, and may beused to characterize molecular size and/or shape, or to separate asample into its components. Suitable hydrodynamic methods may includechromatography, sedimentation, viscometry, and electrophoresis, amongothers.

Imaging methods may involve any method for visualizing a sample or itscomponents, including optical microscopy and electron microscopy, amongothers.

These and other methods such as luminescence lifetime-based backgroundsubtraction are described in further detail in the patent applicationsand publications listed above under “Cross-References,” which areincorporated herein by reference.

F. Additional Examples

This section describes selected additional aspects of the invention, asrecited in the following numbered paragraphs:

1. A device for transferring fluid between first and second locations,the device comprising a mount, and at least one pin moveably attached tothe mount, each pin having a base portion and a tip portion extendingaway from the base portion, the base portion being configured to beattached to the mount, and the tip portion being configured to retain asubstantially reproducible volume of a fluid when brought into contactwith the fluid for transfer between the first and second locations.

2. The device of paragraph 1, wherein the mount includes an elastomericmaterial.

3. The device of paragraph 1, wherein the mount includes a frame capableof dynamically adjusting the arrangement of the pins.

4. The device of paragraph 3, wherein the frame dynamically adjusts thevertical position of the pins relative to a sample.

5. The device of paragraph 3, wherein the frame dynamically adjusts thehorizontal position of the pins relative to a sample.

6. The device of paragraph 1 further comprising a drive mechanismconfigured to move the mount between the first and second positions.

7. The device of paragraph 1, the device configured to transfer fluid toor from a sample container having a reference fiducial encodinginformation about the sample container, further comprising a readercapable of determining the information encoded in the referencefiducial.

8. The device of paragraph 1, wherein the pin is configured to transferan amount of fluid equal to one or fewer microliters.

9. The device of paragraph 1, the device including at least three pins,wherein the pins are arranged in a regular array.

10. The device of paragraph 9, wherein the array is one-dimensional.

11. The device of paragraph 9, wherein the array is two-dimensional.

12. A device for dispensing fluid to a plurality of sample sites in asample holder, the device comprising (a) a dispense site configured tosupport a sample holder having a plurality of sample wells, (b) adispense manifold having a plurality of dispense elements, each dispenseelement capable of dispensing fluid to a sample site, (c) a controllerconfigured to receive information regarding sample positions and toautomatically adjust the effective separation between the dispenseelements to correspond to the separation between the sample site in thesample holder, and (d) a registration device configured to bring thedispense elements and at least a portion of the sample site intoregister, so that fluid may be dispensed from the dispense elements tothe sample sites.

13. The device of paragraph 12, wherein the dispense elements areconfigured for noncontact fluid dispensing.

14. The device of paragraph 12, wherein the sample holder is amicroplate and the sample sites correspond to wells in the microplate.

15. The device of paragraph 12, wherein the sample holder is asubstantially planar surface, and wherein the sample sites arepositioned in the substantially planar surface.

16. The device of paragraph 12, the sample holder having a bar codeencoding information regarding positions of sample sites, furthercomprising a bar code reader configured to scan the bar code.

17. The device of paragraph 12, the sample holder having a referencefiducial encoding information regarding positions of sample sites in thesample holder, further comprising a reference fiducial reader configuredto read the reference fiducial.

18. The device of paragraph 12 further including an imaging deviceconfigured to image at least a portion of the sample holder, so that thepositions of sample sites may be determined.

19. The device of paragraph 12, the sample sites and dispense elementsbeing fixed, wherein the effective separation between the dispenseelements may be adjusted to correspond to the separation between thesample sites in the sample holder by changing the relative orientationof the dispense elements and sample sites.

20. The device of paragraph 19, the dispense elements forming asubstantially linear array constrained to rotate about a pivot point,wherein the relative orientation of the sample sites and dispenseelements may be changed by rotating the dispense elements about thepivot point.

21. The device of paragraph 12 further comprising dispensing fluid fromthe dispense elements to the sample sites.

22. The device of paragraph 21, wherein the fluid is dispensedsimultaneously from each dispense element.

23. The device of paragraph 21, wherein the fluid is dispensedsequentially from each dispense element.

24. The device of paragraph 12, wherein the separations between dispenseelements are variable.

25. The device of paragraph 12, wherein the separations between dispenseelements are fixed.

26. The device of paragraph 12, wherein the dispense elements areconfigured to dispense a range of fluid volume including volumes of lessthan about 1 microliter.

27. A method for dispensing fluid to a plurality of sample sites in asample holder, the method comprising (a) providing a fluid dispenserhaving a plurality of dispense elements, each dispense elementconfigured to dispense fluid, (b) providing a sample holder having aplurality of sample sites, each sample site configured to hold a fluid,(c) obtaining information regarding the positions of the sample sites inthe sample holder, (d) automatically adjusting the effective positionsof the dispense elements to correspond to the positions of the samplesites using the information regarding the positions of the sample sites,(e) bringing the dispense elements and at least a portion of the samplesites into register, and (f) dispensing fluid from the dispense elementsto the sample sites.

28. The method of paragraph 27, wherein the step includes the step ofseparating droplets from the dispense elements without contacting thedroplets to a surface.

29. The method of paragraph 27, wherein the sample holder is amicroplate.

30. The method of paragraph 27, wherein the sample holder has asubstantially planar surface, and wherein the sample sites arepositioned in the substantially planar surface.

31. The method of paragraph 27, wherein the step of obtaininginformation includes scanning an area on the sample holder.

32. The method of paragraph 31, wherein the pre-encoded information isencoded using a bar code, and wherein the sample is scanned using a barcode reader.

33. The method of paragraph 27, wherein the step of obtaininginformation includes measuring the positions of the sample sites duringor immediately prior to dispensing fluid.

34. The method of paragraph 33, wherein the step of measuring thepositions includes the step of imaging at least a portion of the sampleholder using an imaging device.

35. The method of paragraph 33, wherein the step of measuring thepositions includes the steps of locating the position of a referencefiducial and inferring the positions of the sample sites from theposition of the reference fiducial by at least one of interpolation andextrapolation.

36. The method of paragraph 27, the sample sites and dispense elementsbeing fixed, wherein the step of automatically adjusting the effectivepositions of the dispense elements includes the step of changing therelative orientation of the sample sites and dispense elements.

37. The method of paragraph 36, the dispense elements forming asubstantially linear array constrained to rotate about a pivot point,wherein the step of changing the relative orientation of the samplesites and dispense elements includes the step of rotating the dispenseelements about the pivot point.

38. The method of paragraph 27 further comprising dispensing fluid fromthe dispense elements to the sample sites.

39. The method of paragraph 38, wherein the fluid is dispensedsimultaneously from each dispense element.

40. The method of paragraph 38, wherein the fluid is dispensedsequentially from each dispense element.

41. The method of paragraph 27 further comprising (a) providing a secondsample holder having a plurality of sample sites, each sample siteconfigured to hold a fluid, (b) obtaining information regarding thepositions of the sample sites in the second sample holder, and (c)automatically readjusting the effective positions of the dispenseelements to correspond to the positions of the sample sites in thesecond sample holder using the information regarding the positions ofthe sample sites in the second sample holder.

42. The method of paragraph 41, wherein the separations between dispenseelements are variable.

43. The method of paragraph 27, wherein the separations between dispenseelements are fixed.

44. The method of paragraph 27, wherein the dispense elements arecapable of dispensing a fluid aliquot of less than about 1 microliter.

45. A device for spacing microplates, the device comprising a spacingmember dimensioned to maintain separation between stacked first andsecond microplates.

46. The device of paragraph 45, wherein the spacing member includes alid portion.

47. The device of paragraph 46, wherein the lid portion substantiallycovers the sample wells in a microplate stacked below.

48. The device of paragraph 45, wherein the spacing member has a frameportion.

49. The device of paragraph 48, wherein the frame portion substantiallymimics a frame portion of a typical microplate so that a stacker ordestacker can manipulate the spacing member as it would a microplate.

50. The device of paragraph 48, wherein the frame portion has at leastone aperture for allowing environmental circulation between adjacentlystacked microplates.

51. The device of paragraph 45, wherein the spacing member includes oneor more upwardly extending projections.

52. The device of paragraph 46, wherein the lid portion has at least oneaperture for allowing environmental access to samples contained in wellsunder the spacing member.

53. A method of allowing environmental access between stackedmicroplates, the method comprising spacing adjacent microplates in astack.

54. The method of paragraph 53 further comprising the step of providingat least one aperture in a side of a frame member between stackedmicroplates.

55. The method of paragraph 53 further comprising the step ofcontrolling ambient gas constituents and temperature around and betweenstacked microplates.

56. The method of paragraph 53 further comprising the step ofcirculating a gas between stacked microplates.

57. A method of allowing environmental access between stackedmicroplates, the method comprising projecting spacing members betweenmicroplates in a stack.

58. A method of allowing environmental access between stackedmicroplates, the method comprising elevating one microplate aboveanother microplate by situating a spacing member between themicroplates.

59. The method of paragraph 58 further comprising the step ofconfiguring the spacing member to mimic the perimetral dimensions of atypical microplate so that the spacing member can be manipulated by amicroplate stacker or destacker.

60. The method of paragraph 59 further comprising the step ofautomatically stacking and de-stacking the spacing member.

61. A microplate comprising (a) a frame member, (b) a plurality ofsample wells contained within the frame member, the wells having upperedges contained in a common plane, and (c) one or more projectionsextending above the plane, so that the wells maintain a spacedrelationship to a microplate stacked on top.

62. A system for automatically covering and uncovering wells in amicroplate, the system comprising (a) a supply of flexible sheetmembers, (b) a sealing device that automatically positions a sealingsheet over an array of wells in a microplate, and presses the sealingsheet onto the microplate so that the wells are substantially sealed,and (c) a sheet removal device that automatically contacts and lifts thesealing sheet off the microplate.

63. The system of paragraph 62, wherein the sheet removal device has apicking member that removes the sealing sheet by gripping an edge of thesheet.

64. The system of paragraph 62, wherein the sheet removal device has apicking member that pierces and then lifts the sheet from themicroplate.

65. The system of paragraph 62, wherein the sheet removal device has apicking member that applies a vacuum to at least a portion of the sheet.

66. The system of paragraph 62, wherein the sheet removal device has apicking member that applies an adhesive to at least a portion of thesealing sheet.

67. A device for removing a sealing sheet from a microplate, the devicecomprising (a) a microplate-sheet removal station, and (b) asheet-handling mechanism positioned near the sheet removal station, andconfigured to contact and lift a sealing sheet off a sealed microplate.

68. The device of paragraph 67, wherein the sheet-handling mechanism hasa picking member that grips an edge of the sheet.

69. The device of paragraph 67, wherein the sheet-handling mechanism hasa picking member that pierces the sheet and then lifts the sheet fromthe microplate.

70. The device of paragraph 67, wherein the sheet-handling mechanism hasa picking member that applies a vacuum to at least a portion of thesheet.

71. The device of paragraph 67, wherein the sheet-handling mechanism hasa picking member that applies an adhesive to at least a portion of thesheet.

72. A microplate sealing system comprising (a) a first microplate havinga plurality of wells in a sample containment area, and a frame areasurrounding the sample containment area, wherein the microplate isdesigned so that it can be stacked below a second microplate, and (b) aflexible sealing sheet covering the sample containment area of the firstmicroplate without contacting the second microplate when it is stackedon top of the first microplate.

73. The system of paragraph 72, wherein the first microplate has atleast one recess in a side exposing top and bottom sides of an edge ofthe sealing sheet for easy gripping and removal of the sheet from themicroplate.

74. The system of paragraph 72, wherein the flexible sealing sheet isdimensioned to substantially cover the sample containment area of themicroplate without contacting the frame area of the microplate.

75. The system of paragraph 72, wherein the flexible sealing sheet issubstantially optically transparent relative to an optical analysis tobe carried out on a sample contained in a well of the microplate.

76. The system of paragraph 72, wherein the flexible sealing sheet issubstantially optically opaque.

77. A cover material for microplates comprising (a) a continuous roll ofbacking material, and (b) a series of discrete sealing sheets releasablyfixed on a surface of the backing material, wherein each sheet isdimensioned to cover substantially all of a plurality of wells of astandard microplate while leaving a peripheral top portion of themicroplate uncovered.

78. A method of applying a cover sheet to a microplate, the methodcomprising (a) applying a sealing sheet over substantially all of thewells in a microplate without covering a continuous perimetral topregion of the microplate, and (b) exposing top and bottom sides of anedge portion of the sealing sheet for gripping when the sealing sheet isremoved.

79. The method of paragraph 78, wherein the applying step is performedmanually.

80. The method of paragraph 78, wherein the applying step is automated.

81. The method of paragraph 78 further comprising the step of removingthe sealing sheet from the microplate prior to performing an analysis ona sample contained in a well in the microplate.

82. The method of paragraph 81, wherein the removing step is performedmanually.

83. The method of paragraph 81, wherein the removing step is automated.

84. The method of paragraph 78 further comprising the step of forming atleast one recess in a side of the microplate so that top and bottomsides of the edge portion are exposed.

85. The method of paragraph 78, wherein the sealing sheet issubstantially rectangular.

86. A sample holder comprising (a) a top portion containing an array ofsample wells, and (b) a seal covering one or more of the wells, whereintop and bottom sides of an edge portion of the seal are exposed tofacilitate removal of the seal.

87. The sample holder of paragraph 86, wherein the top portion has atleast one recess below an edge of the seal.

88. The sample holder of paragraph 86, wherein the top portion has aplurality of recesses on at least two sides of the plate.

89. The sample holder of paragraph 86, wherein the sample holder is amicroplate.

90. The sample holder of paragraph 86, wherein the seal is substantiallyrectangular.

91. A sample holder comprising a top portion containing an array ofsample wells within a perimeter portion, wherein the top portion has atleast one recess in the perimeter portion for exposing an edge of a sealthat covers one or more of the wells.

92. An automated device comprising a gripping mechanism configured tocontact an exposed edge of a sealing sheet on a microplate, and toremove the sheet from the microplate.

93. The automated device of paragraph 92, wherein the gripping mechanismpierces and lifts the sealing sheet from the microplate.

94. The automated device of paragraph 92, wherein the gripping mechanismapplies a vacuum to at least a portion of the sealing sheet.

95. The automated device paragraph 92, wherein the gripping mechanismapplies an adhesive to at least a portion of the sealing sheet.

96. A first sample container device comprising (a) a base portion, (b) aplurality of wells formed in a top side, the wells having upper edgesdefining a plane above the base portion, and (c) at least one elevationmechanism extending above the plane to hold a second sample container inspaced relation to the first sample container.

97. The device of paragraph 96, wherein the elevation mechanism includesat least four post members connected to the top side and extendingupward from the plane.

98. The device of paragraph 96, wherein the elevation mechanism includesfour post members, each post member extending upward from a corner ofthe first sample container.

99. The device of paragraph 96, wherein the elevation mechanism isindependent from the first sample container.

100. The device of paragraph 96, wherein the base portion has at leastone aperture to allow gas circulation under the wells.

101. The device of paragraph 196, wherein wells are provided in the topside of the first sample container in a density of at least about 4wells per 81 mm².

102. The device of paragraph 196, wherein the elevation mechanism has alid portion that substantially covers all of the wells in the top sideof the first sample container.

103. An incubation system, comprising (a) an enclosure, (b) at least twomicroplates stacked within the enclosure, each microplate having aplurality of wells for containing samples, and (c) a spacing mechanismbetween the two plates to allow gas diffusion and thermal equilibrationaround samples contained in wells of the microplate.

104. The system of paragraph 103, wherein the spacing mechanism has anouter frame dimension similar to a microplate so that a stacking devicedesigned to handle microplates can also handle the spacing mechanism.

105. The system of paragraph 103, wherein at least one of themicroplates has a lateral aperture for allowing gas to circulate betweenthe plates.

106. The system of paragraph 103, wherein the spacing mechanism isformed in a top side of one of the microplates.

107. The system of paragraph 103, wherein the spacing mechanism is aseparate piece from the two microplates.

108. The system of paragraph 103, wherein the enclosure is a room.

109. The system of paragraph 103, wherein the enclosure is a sealedchamber.

110. The system of paragraph 103, wherein the enclosure has a valve forallowing controlled passage of gas in and out of the enclosure.

111. A method of controlling a gas environment around a plurality ofsamples, comprising (a) dispensing samples into a plurality of wells ina first microplate and a second microplate, (b) stacking a spacingmechanism on top of the first microplate, and (c) stacking the secondmicroplate on top of the spacing mechanism to allow gas diffusion andthermal equilibration around the samples.

112. The method of paragraph 111 further comprising the step ofproviding lateral apertures in the microplates.

113. The method of paragraph 111 further comprising the step ofmonitoring the temperature of a space containing the microplates.

114. The method of paragraph 111 further comprising the step ofincubating the microplates in an enclosure.

115. A device for spacing microplates, the device comprising (a) firstand second microplates, and (b) a spacing member separating the firstand second microplates in a stack.

116. The device of paragraph 115, wherein the spacing member includes alid portion.

117. The device of paragraph 115, wherein each microplate has aplurality of sample wells, the lid portion substantially covering thesample wells so that evaporation from the sample wells is minimizedwhile allowing thermal communication between the environment and thespace between the microplates.

118. The device of paragraph 115, wherein the spacing member has a frameportion, the frame portion being designed to substantially mimic a frameportion of a typical microplate so that a stacker or destacker canmanipulate the spacing member as it would a microplate.

119. The device of paragraph 118, wherein the frame portion has at leastone aperture for allowing gas and thermal circulation between themicroplates.

120. The device of paragraph 115, wherein the spacing member includesone or more upwardly extending projections.

121. The device of paragraph 114, wherein the lid portion has at leastone aperture for allowing environmental access to samples contained inwells under the spacing member.

Although the invention has been disclosed in preferred forms, thespecific embodiments thereof as disclosed and illustrated herein are notto be considered in a limiting sense, because numerous variations arepossible. Applicants regard the subject matter of their invention toinclude all novel and nonobvious combinations and subcombinations of thevarious elements, features, functions, and/or properties disclosedherein. No single feature, function, element or property of thedisclosed embodiments is essential. The following claims define certaincombinations and subcombinations of features, functions, elements,and/or properties that are regarded as novel and nonobvious. Othercombinations and subcombinations may be claimed through amendment of thepresent claims or presentation of new claims in this or a relatedapplication. Such claims, whether they are broader, narrower, or equalin scope to the original claims, also are regarded as included withinthe subject matter of applicant's invention.

1. A system for delivering fluid to a sample holder comprising a fluidsource, a positive displacement pump connected to the fluid source, adispenser assembly having an orifice, and a conduit path extending fromthe pump to the orifice of the dispenser assembly, wherein operation ofthe positive displacement pump provides non-contact deposition of fluidaliquots having a volume of less than about 5 microliters per aliquotwithout closing or constricting the conduit path between the depositionof successive fluid aliquots.
 2. The system of claim 1 furthercomprising a controller that determines the volume of each fluidaliquot.
 3. The system of claim 1, wherein the pump pumps incrementallyat a rate corresponding to the rate of aliquot deposition.
 4. The systemof claim 1, wherein the dispenser assembly has a hydrophobic tipportion.
 5. The system of claim 4, wherein the tip portion is made of aheat-shrinkable material.
 6. The system of claim 5, wherein the tipportion is made of material selected from the group consisting of PTFE,polypropylene, polyethylene, and FEP.
 7. The system of claim 1, whereinthe dispenser assembly has a tip portion made of sapphire.
 8. The systemof claim 1, wherein the orifice is formed at an end of a tube-like tipportion, the tip portion having a wall thickness around the orifice ofless than about 8 thousandths of an inch.
 9. The system of claim 1,wherein the orifice has an inner diameter of less than about 200microns.
 10. The system of claim 8, wherein the pump is connected to thedispenser assembly by a tube having a distal end, the tip portion havinga flange on a proximal end, the distal end of the tube being held incontact with the flange of the tip portion.
 11. The system of claim 1,wherein the pump is a syringe pump.
 12. The system of claim 11, whereinthe syringe pump has a linear motor.
 13. The system of claim 11, whereinthe pump has a stepper motor.
 14. The system of claim 10, wherein thedispenser assembly includes a manifold for holding the distal end of thetube in contact with the flange of the tip portion.
 15. The system ofclaim 14, wherein the same manifold secures connection of a plurality oftubes to respective tip portions to define an array of fluid dispensingchannels.
 16. The system of claim 15, wherein the array of fluiddispensing channels corresponds to an array of wells in a microplate.17. The system of claim 15, wherein the array of fluid dispensingchannels corresponds to an array of sites on a biochip.
 18. The systemof claim 15, wherein the array corresponds to wells in a standard96-well microplate.
 19. The system of claim 15 further comprising asample holder registration mechanism that alters the position of asample holder relative to the manifold so that the same array of fluiddispensing channels can be used to deposit fluid into sample holdershaving varying densities of deposition sites.
 20. A fluid dispensingsystem comprising an array of at least eight dispense tips, eachdispense tip being connected to a separate syringe pump, a fluid sourcebank, the fluid source bank having plural fluid reservoirs, and achangeable fluid conduit network capable of permitting: (a) each of atleast eight of the pumps to be connected to a separate fluid reservoir,(b) each of at least eight of the pumps to be connected to the samefluid reservoir, and (c) any subset of pumps to be connected to the samefluid reservoir while one or more other pumps are connected to anotherfluid reservoir, wherein the dispense tips are configured to dispensedroplets in a range of volumes less than about 5 microliters per dropletwithout contacting the droplets to a surface.
 21. The system of claim20, wherein each dispense tip has a hydrophobic wall defining an orificehaving a diameter of less than about 200 microns, the wall having athickness of less than about 8 thousandths of an inch.
 22. The system ofclaim 20, wherein the dispense tips are made of a material selected fromthe group consisting of sapphire, PTFE, polypropylene, polyethylene, andFEP.
 23. The system of claim 20, wherein each pump includes a linearmotor.
 24. A device for dispensing fluid to a sample or sample holder,the device comprising a fluid reservoir, a syringe pump device connectedto the fluid reservoir, and a dispense element operatively connected tothe pump device, wherein the pump device drives fluid incrementally tothe dispense element with sufficient velocity and acceleration so that afluid aliquot of less than about five microliters separates from thedispense element without contacting the sample or the sample holder. 25.The device of claim 24, wherein the dispense element has a hydrophobictip with a tube-like wall defining an orifice and is configured toreduce the affinity of dispensed fluid for the dispense element, so thatdispensed fluid separates from the dispense element.
 26. The device ofclaim 25, wherein the wall of the tip has a thickness of less than about8 thousandths of an inch.
 27. The device of claim 25, wherein theorifice has a diameter of less than about 200 microliters.
 28. Thedevice of claim 24, wherein the dispense element has a tip portion madeof a material selected from the group consisting of PTFE, polypropylene,polyethylene, and FEP.